# Screening of antimicrobial activity in venom: Exploring key parameters

Awatef Ouertani, Chloé Mollet, Yasmine Boughanmi, Harold de Pomyers, Amor Mosbah, Hadda-Imene Ouzari, Ameur Cherif, Didier Gigmes, Marc Maresca, Kamel Mabrouk

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## To cite this version:

Awatef Ouertani, Chloé Mollet, Yasmine Boughanmi, Harold de Pomyers, Amor Mosbah, et al. Screening of antimicrobial activity in venom: Exploring key parameters. Toxicon, 2024, 251, 10.1016/j.toxicon.2024.108135. hal-04951847

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# Screening of antimicrobial activity in venom: Exploring key parameters

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Awatef Ouertani<sup>a,1</sup>, Chloé Mollet<sup>b,c,1</sup>, Yasmine Boughanmi<sup>b,c</sup>, Harold de Pomyers<sup>c</sup>,
Amor Mosbah<sup>a</sup>, Hadda-Imene Ouzari<sup>d</sup>, Ameur Cherif<sup>a</sup>, Didier Gigmes<sup>b</sup>, Marc Maresca<sup>e</sup>,
Kamel Mabrouk<sup>b,*</sup>

## ARTICLE INFO

Handling editor: Denise Tambourgi

**Keywords:**

Antimicrobial resistance

Venoms

New molecules

Screening method

Influential factors

## ABSTRACT

The escalating challenge of antibiotic resistance significantly threatens global health, underscoring the critical need for new antimicrobial agents. Venoms, increasingly recognized as reservoirs of bioactive compounds with diverse pharmacological effects, have been the focus of recent research. This work evaluates the use of various screening methodologies in assessing the antimicrobial activities of 185 venoms against some gram positive and gram negative bacteria, including *E. coli* ATCC 8739, *B. subtilis* ATCC 6633, *P. aeruginosa* ATCC 9027, and *S. aureus* ATCC 6538P species and explores the influence of settings on the findings. Furthermore, the research explored the possibility of purifying antimicrobial molecules from venoms through HPLC. Several fractions demonstrated antimicrobial activity against the tested strains.

Our results reveal that the measured antimicrobial efficacy of venoms varies according to: i) venom concentration, ii) the detection method, including microdilution and radial diffusion assays, and iii) the choice of culture medium, specifically LB or MH.

This strategy has allowed us, for the first time, to identify antimicrobial activity in: i) *Bitis arietans* venom against *P. aeruginosa* ATCC 9027, ii) *Naja nubiae* and *Bothrops lanceolatus* against *B. subtilis* ATCC 6633, *P. aeruginosa* ATCC 9027, and *S. aureus* ATCC 6538P, and iii) *Hadogenes zuluanus*, *Mesobuthus caucasicus*, *Nebo hierichonticus*, *Opistophthalmus wahlbergii* scorpions, and *Mylabris quadripunctata* beetles against *S. aureus* ATCC 6538P.

These findings highlight venoms potential as effective antimicrobial resources and improve our understanding of key factors critical for an accurate detection of venoms antimicrobial properties.

## 1. Introduction

Antimicrobial resistance (AMR) is a critical global health threat that continues to escalate (Nikaido, 2009). Microbes including bacteria, viruses, fungi and parasites can adapt over time, leading to antimicrobial resistance (Larsson and Flach, 2022). As a result, drugs that were initially effective in treating infections caused by these microbes may become ineffective. This challenges infections treatment strategies and increases the risk of disease transmission. Additionally, routine medical procedures such as surgery may become riskier, as patients are more susceptible to severe illness and even death due to AMR (Magiorakos et al., 2012).

The combination of increasing resistance and limited investment in new antibiotics discovery and development has left healthcare providers with few options for treating multi-drug-resistant bacterial infections (Morehead and Scarbrough, 2018). Gram-negative bacteria, such as *Pseudomonas aeruginosa*, *Salmonella* sp., and other members of the Enterobacteriaceae, as well as Gram-positive species like *Staphylococcus aureus*, are recognized as pathogens with limited treatment options due to their resistance to available antibiotics (World Health Organization, 2017). Identifying additional unique bioactive molecules is crucial as the majority of antibiotics approved in the last decade are ineffective against the most concerning pathogens

<footnote><sup>a</sup> Univ. Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, Tunisia</footnote>

<footnote><sup>b</sup> Aix Marseille Univ, CNRS, ICR, UMR 7273, 13397, Marseille, Cedex 20, France</footnote>

<footnote><sup>c</sup> LATOXAN SAS, 845 avenue Pierre Brossolette, 26800, Portes-les-Valence, France</footnote>

<footnote><sup>d</sup> Université Tunis El Manar, FST, LMBA (LR03ES03), 2092, Campus Universitaire, Tunis, Tunisia</footnote>

<footnote><sup>e</sup> Aix Marseille Univ, CNRS, Centrale Marseille, ISM2, 13013, Marseille, France</footnote>

<footnote>* Corresponding author. Aix Marseille University, CNRS, ICR UMR 7273, 13397, Marseille, Cedex 20, France.
*E-mail address:* kamel.mabrouk@univ-amu.fr (K. Mabrouk).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
including carbapenem-resistant Gram-negative bacteria (Coppola et al., 2022). Natural products continue to offer promising avenues for novel therapeutics. Nature has been a traditional source and inspiration for drug discovery for thousands of years (Rossiter et al., 2017), among which animal poisons and venoms, rich in proteins, peptides, neurotransmitters, and other compounds, represent a promising source of potential therapeutics for both human and veterinary medicine (Hurka et al., 2022). These components are extensively studied for a wide range of pharmacological properties. The use of natural products in general, and venoms in particular, can be constrained by their scarcity and high production costs. However, identifying medium-sized peptides (<50 amino acids) and synthesizing them either chemically or through expression in unlimited quantities may address these limitations. Indeed, advancements in biotechnology and synthetic biology provide promising avenues for the scalable production of these compounds, potentially reducing costs and enhancing accessibility. This approach ensures that the therapeutic potential of bioactive peptides can be effectively harnessed, despite the initial rarity of their natural sources. However, it is notable that relatively little is known about the antimicrobial activity of venoms (Alam et al., 2019).

Numerous techniques are available to assess the *in vitro* antimicrobial activity of extracts or pure compounds. Commonly used methods include disk diffusion, broth dilution, or agar dilution methods. With the growing interest in novel antimicrobial products capable of combating multidrug resistant bacteria, it is essential to better understand the existing methods used to screen the antimicrobial potential of extracts or pure molecules, particularly in the context of human health and veterinary science (Balouiri et al., 2016).

The objective of this research was to appraise the antimicrobial activities of over 185 animal venoms on a range of selected bacteria. This work aimed at comparing the weight of experiment variables, including the screening method, venom concentration, and the choice of culture media, on the measurement of antimicrobial activity of venoms.

## 2. Material and methods

### 2.1. Venoms

A collection of lyophilized venoms of snakes, scorpions, spiders and other venomous animals was provided by Latoxan (Portes-lès-Valence, France) and stored at 4 °C. The initial stocks of venoms were prepared in HPLC-grade water (34877-2.5L-M; Sigma-Aldrich, Saint-Louis, MO, USA) or Milli-Q water at a concentration of 10 mg/mL. Each solution was clarified by centrifugation (5 min at 5000 rpm). To preserve enzymatic activity, all venom supernatants were stored at -80 °C until use. Repeated freeze-thaw cycles were avoided by aliquoting the samples. Venoms were handled on ice.

### 2.2. Antimicrobial activity assay of venoms

#### 2.2.1. Microbroth dilution method

The venoms were initially screened using a liquid bacterial growth inhibition test in 96-well plates (Cat. No. 10062-900, VWR, Radnor, PA, USA). The test was performed using non-pathogenic reference strains: Gram positive, *Bacillus subtilis* (B. subtilis) ATCC 6633, B. as well as Gram negative *E. coli* ATCC 8739. Pathogenic reference strains: Gram positive, *Staphylococcus aureus* ATCC 6538P and Gram negative *Pseudomonas aeruginosa* ATCC 9027. Each venom was tested in duplicate on two independent experiments. On Day 1, test strain was inoculated from a cryotube onto Lysogeny Broth (LB) agar medium and incubated at 37 °C for 24 h. On Day 2, one Colony Forming Unit (CFU) of each test strain was cultured overnight (between 20 and 22h) in 2 ml of LB medium at 37 °C, with shaking at 200 rpm (Unitron HT Infors, Bottmingen, Switzerland). On day 3, each well containing 40 µL of sample (whole venom, fractions, sub-fractions) was supplemented with 160 µL of the bacterial strain tested, previously diluted in LB medium to a final

concentration of 1,10⁵ to 1,10⁶ CFU/ml. Whole venoms were tested at 10 mg/ml from a 50 mg/ml stock solution. Some venoms were tested at 1 and 0.1 mg/ml from stock solutions of 5 and 0.5 mg/ml, respectively. 40 µL of the antibiotic amoxicillin (stock solution of 250 µg/ml; Cat. No. A8523-25G, Sigma-Aldrich, St. Louis, MO, USA) for *B. subtilis*, *E. coli*, and *S. aureus*, or gentamicin (at 250 µg/l; Cat. No. SV30080.01, HyClone TM, San Angelo, Texas, USA) for *P. aeruginosa*, was added to 160 µL of the bacterial suspension as positive control. Sterile Milli-Q water was used as a negative control following the same procedure. Each point was run in duplicate on the same plate, and each condition was repeated in at least two different experiments. On day 4, inhibition of bacterial growth was assessed by measuring absorbance (optical density) at 360 nm using a microplate reader (PowerWave X340, BioTek Instruments, Winooski, VT, USA) after 17-18 h of incubation at 37 °C (OD reading software Gen5 2.05, BioTek Instruments, Winooski, VT, USA) (Olleik et al., 2019).

#### 2.2.2. Radial diffusion assay

A collection of 185 venoms was tested for their antimicrobial activities against *Staphylococcus aureus* ATCC 6538P and *Pseudomonas aeruginosa* ATCC 9027 using the radial diffusion assay method (Lehrer et al., 1991). Briefly, on Day 1, each test strain was inoculated from a cryotube onto LB agar medium and incubated at 37 °C for 24 h. On day 2, one CFU of each test strain was cultured overnight in 2 ml LB medium at 37 °C, with shaking at 200 rpm (Unitron HT Infors, Bottmingen, Switzerland). After 17-18 h of incubation, 75 µL of the saturated culture was transferred to 15 mL LB medium and incubated for 2-2.5 h at 37 °C, with shaking at 200 rpm. The bacterial suspension was then centrifuged for 5 min at 2000 rpm. The bacterial pellet was washed with 10 mL PBS 1X (Cat. No. 10010-023, Gibco®, Carlsbad, CA, USA) at 4 °C by centrifugation (5 min, 2000 rpm, 4 °C). The pellet was then resuspended in 5 or 10 ml PBS to achieve an OD max not exceeding 1. Absorbance of bacterial solutions was measured at 620 nm from a 1 mL aliquot. The volume required to obtain 4.10⁶ CFU/mL was determined using the following calculation: A620nm x 2.5.10⁸ CFU/mL = CFU/m. Ten milliliters of Underlay gel maintained at 42 °C was inoculated with 4.10⁶ CFU/mL in a petri dish (120x120 or ø = 90 mm) on a level surface and placed at 4 °C for 10 min for perfect gelation. Wells were sterilely hollowed out using a punch (ø = 4 mm or 6 mm). Crude venoms were tested at 100 µg (from a 10 mg/mL solution). After allowing diffusion for 10 min on the bench, the dish was incubated for 3 h at 37 °C. Each Underlay gel was then covered with 10 mL of Overlay gel. After complete gelation for 15 min at 4 °C, the petri dishes were inverted and incubated at 37 °C overnight. After 17-18 h of incubation, growth inhibition was examined by measuring the diameter of the inhibition halos formed (in millimeters), including the diameter of the 4 or 6 mm well. To present the results, the diameter of the wells was subtracted from the inhibition halos obtained.

### 2.3. Comparative research

A comparative analysis between the radial diffusion assay and the microbroth dilution method was carried out using *S. aureus* ATCC6538P and *P.aeruginosa* ATCC 9027. Venoms activities were tested using both techniques and LB medium.

The venom concentrations used in the microbroth dilution method, and the radial diffusion assay (RDA) vary due to the inherent differences in sensitivity between these assays. The RDA is more sensitive and can detect antimicrobial activity at lower concentrations because the antimicrobial agents diffuse into a localized area within the agar medium (Lehrer and Barton, 1987). Therefore, we used a venom concentration of 100 µg in the RDA. In contrast, the microbroth dilution method requires higher concentrations to achieve detectable inhibition in a liquid medium, leading us to use a venom concentration of 10 mg/ml (40 µL means 400 µg). This approach ensures the optimal detection of antimicrobial activity in each assay.
## 2.4. Antimicrobial molecule purification using high performance liquid chromatography HPLC

The selected venom solutions (50 mg venom/10 ml HPLC water) were centrifuged (5min/5000 rpm) then filtered through 0,45 µmfilter. Fractionation of venom solutions was performed using preparative HPLC (Agilent 1260 Infinity Agilent Technologies, Santa Clara, CA, USA) set with a quaternary gradient pump system and a multi-wavelength UV detector (Multi-Wavelength Detector = MWD). A preparative C18 column (250–21 mm, particles 5 µm, pores 100 Å) from MACHERY NAGEL was used. Acetonitrile 0.1% TFA (solvent B) served as the mobile phase, with a flow rate of 6 ml/min. Elution of the different peaks was performed with 60% acetonitrile over 60 min. Peaks associated with active molecules were lyophilized, dissolved in distilled water and tested for antimicrobial activity using microbroth dilution method (Verdoni et al., 2016; Chalier et al., 2020).

# 3. Results and discussion

## 3.1. Screening of antimicrobial activity of venoms

### 3.1.1. Screening of venoms using microbroth dilution method

Using microbroth dilution method, 185 venoms were screened for their antimicrobial properties against both non-pathogenic reference strains (B. subtilis ATCC 6633 and E. coli ATCC 8739) and pathogenic ones Staphylococcus aureus ATCC 6538P and Pseudomonas aeruginosa ATCC 9027 at a final concentration of 10 mg/ml. The results indicate that 68%, 55%, 57%, and 61% of the tested venoms showed activity against B. subtilis, E. coli, S. aureus and P. aeruginosa, respectively (Fig. 1). Notably, pathogenic strains exhibited a higher level of resistance to venom activity compared to non-pathogenic strains, as evidenced by an inhibition rate of 60.5%.

Among the venoms tested, only 5% exhibited activity exclusively against pathogenic strains, with 56% of those specifically inhibiting S. aureus. This finding is significant as an effective antibiotic, or anti-bacterial agent should, target pathogenic strains while sparing commensal strains (Helmy et al., 2023). Furthermore, only 40% of the tested venoms demonstrated broad-spectrum antibacterial activity inhibiting the growth of both pathogenic and non-pathogenic Gram-negative and Gram-positive strains at a final concentration of 10 mg/mL. Notably, all venoms displaying broad spectrum activity were derived from snakes, which constituted the most represented family in the research, with 141 samples accounting for 98% of the venoms tested for antibacterial properties. It is noteworthy that venoms belonging to the Viperidae family (Crotalinae and Viperinae) were more effective than those from the Elapidae family.

Results indicated that spider venoms did not exhibit antibacterial activity against the tested strains. Only a small percentage (15%) of

amphibian skin secretions, insect, and scorpion venoms inhibited the bacterial growth of non-pathogenic strains. However, venom derived from amphibian skin secretions proved effective against S. aureus ATCC 6538P (Fig. 2). According to various researches, venoms of amphibian and arthropods are a rich source of antimicrobial molecules. In a recent work, a novel antimicrobial peptide, dendropsophin 1, was purified from the skin secretion of the endemic Colombian frog Dendropsophus columbianus (Triana-Vidal et al., 2018).

Venoms inhibiting the growth of B. subtilis ATCC 6633 and E. coli ATCC 8739 were further diluted and tested at lower concentrations of 1 mg/ml and 0.1 mg/ml to assess if they maintained their activity. The findings revealed that at a final concentration of 1 mg/ml, only 46% and 31% continued to exhibit activity against B. subtilis ATCC 6633 and E. coli ATCC 8739, respectively. Among the 46% of venoms active against B. subtilis ATCC 6633 only 22% remained active at a final concentration of 0.1 mg/ml. These variations in antimicrobial effectiveness underscore the significance of the chosen venom concentration for testing. The observed variations could be attributed to the differential presence of active molecules in the venom at varying dilutions.

Snake venoms were found to be particularly effective: they comprised approximately 98% of the crude active venoms against B. subtilis ATCC 6633 and E. coli ATCC 8739 at a final concentration of 1 mg/ml. From the venoms that inhibited the growth of pathogenic strains at a final concentration of 10 mg/ml, only thirty were further tested at a final concentration of 1 mg/mL. The results indicated that 97% retained their activity against S. aureus ATCC 6538P and 88% against P. aeruginosa ATCC 9027.

Some venoms have been effective against S. aureus ATCC 6538P and B. subtilis ATCC 6633 at a final concentration of 10 mg/ml, but their efficacy diminished at lower concentrations of 1 mg/mL. However, these venoms remained active against S. aureus and P. aeruginosa at both 10 and 1 mg/ml concentrations (Table 1). Notably among these venoms are those from Bitis gabonica rhinoceros, Bothriechis schlegelii and Bothrops atrox. For instance, Samy et al. (2007) reported the antimicrobial activity of Bitis gabonica rhinoceros venom at a concentration of 100 µg/ml, against E. coli and S. aureus using the disc diffusion method, the crude venom was inactive against P. aeruginosa (Perumal Samy et al., 2007). These varied results suggest that an appropriate testing protocol should start with high concentrations and gradually decrease until the activity is lost. Additionally, the findings imply that antibiotics at low concentrations may not be lethal to certain microorganisms (Bernier and Surette, 2013). In another work Bothriechis schlegelii venom was active against S. aureus, P. aeruginosa, B. subtilis and E. coli at a final concentration of 10 mg/ml, but lost its activity against E. coli and B. subtilis at 1 mg/mL. Munoz et al. (2014) demonstrated that 10 µg of Bs LAAO (l-amino acid oxidase), purified from Bothriechis schlegelii venom and tested using the agar diffusion assay, exhibited inhibitory effects against S. aureus with a MIC of 4 µg/ml and against Acinetobacter baumannii with a MIC of

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Proportion of active and inactive venoms at concentration of 10 mg/mL against bacterial strains: B. subtilis (ATCC 6633), E. coli (ATCC 8739), S. aureus (ATCC 6538P) and P. aeruginosa (ATCC 9027) as determined by the microbroth dilution method.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Distribution of venoms inhibiting the growth of *B. subtilis* (ATCC 6633), *E. coli* (ATCC 8739), *S. aureus* (ATCC 6538P) and *P. aeruginosa* (ATCC 9027) at a concentration of 10 mg/mL categorized sub-family. (*) indicates the number of venoms tested within each sub-family.</figcaption></figure>

<caption>**Table 1**</caption>

Effect of venom concentration on the antimicrobial activity determined through liquid bacterial growth inhibition test. *B. subtilis* (ATCC 6633), *E. coli* (ATCC 8739), *S. aureus* (ATCC 6538P) and *P. aeruginosa* (ATCC 9027).

<table><thead><tr><th></th><th>E. coli<br>ATCC<br>8739</th><th>B. subtilis<br>ATCC<br>6633</th><th>P. aeruginosa<br>ATCC 9027</th><th>S. aureus<br>ATCC<br>6538P</th></tr></thead><tbody><tr><td>Venom concentration (mg/mL)</td><td>10</td><td>1</td><td>10</td><td>1</td></tr><tr><td>Agkistrodon contortrix contortrix</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Agkistrodon piscivorus conanti</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Atheris nitschei nitschei</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Atheris squamigera</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Atropoides mexicanus</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Atropoides nummifer</td><td>+</td><td>+</td><td>-</td><td>+</td></tr><tr><td>Bitis caudalis</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Bitis gabonica rhinoceros</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Bitis nasicornis (Burundi)</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Bitis nasicornis (WestAfrica)</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Bothriechis schlegelii</td><td>+</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Bothrops asper (Costa Rica)</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Bothrops asper (Ecuador)</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Bothrops atrox (Brazil)</td><td>+</td><td>-</td><td>-</td><td>+</td></tr><tr><td>Bothrops colombiensis</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>Bothrops lanceolatus adultes</td><td>-</td><td>-</td><td>+</td><td>+</td></tr><tr><td>Virido vipera/Trimeresurus gumprechti</td><td>+</td><td>+</td><td>+</td><td>+</td></tr></tbody></table>

2 µg/mL, but showed no effect against *E.coli* (Muñoz et al., 2014)

Bothrops atrox venom exhibited antimicrobial activity against *S. aureus*, *P. aeruginosa*, *B. subtilis* and *E. coli* at a final concentration of 10 mg/mL. However, its activity diminished against *E. coli* and *B. subtilis* at a lower concentration of 1 mg/mL. Samy et al. (2007) demonstrated that LAAO from *B. atrox* venom had significant inhibitory effects against *S. aureus*, *Proteus mirabilis* at a concentration of 100 µg/ml using disc diffusion method, but showed no activity against *E. coli*, *P. aeruginosa* and *Enterobacter aerogenes* (Perumal Samy et al., 2007). Furthermore, Batroxicidin (BatxC) isolated from *Bothrops atrox* venom exhibited

antimicrobial effects against Gram bacteria (*E. coli* ATCC 25922 at 0.25 µg/ml, *P. aeruginosa* ATCC 27853 at 1 µg/ml, *E. coli* at 16 µg/ml, *P. aeruginosa* at 16 µg/ml, *K. pneumoniae* at 8 µg/ml, *A. baumannii* at 16 µg/ml) and Gram+ bacteria, (*E. faecalis* ATCC 29212 at 32 µg/ml, *S. aureus* ATCC 29213 at 32 µg/ml, *E. faecalis* at 128 µg/ml, *S. aureus* at 32 µg/ml, *S. pyogenes* at 16 µg/ml), as reported by Falcao et al., 2014. Oliveira-Júnior et al. (2018) observed that the MIC of Batroxicidin was promising against ATCC bacteria, particularly against the Multi Drug Resistant Bacteria such as *K. pneumoniae* 1445333, *K. pneumoniae* 1410503, *E. coli* 2101123 and *E. coli* 1812446. Batroxicidin was active against *K. pneumoniae* ATCC 13883 and *E. coli* ATCC 25922 (8 µg/ml for both), and *P. aeruginosa* ATCC 27853 (8 µg/ml). Additionally, Batroxicidin showed activity against clinically isolated strains of *K. pneumoniae*, *E. coli* 2101123, and *E. coli* 1812446 with MICs of 16 µg/ml, 8 µg/ml and 4 µg/ml respectively (Oliveira-Júnior et al., 2018).

Our results largely align with these observations, though some differences are noted. The variability in venoms impact on specific microorganisms could be attributed to the different methodologies employed in testing. Notably, our research utilized crude venom, in contrast to the purified fractions used in other works at lower concentrations. The use of crude extracts, as opposed to isolated molecules, may offer a significant advantage, as the extract could contain various molecules that synergistically interact with the bioactive compound, thereby enhancing its effect, as suggested by Barba-Ostria et al. (2022).

Because of its ease of handling and its ability to be applied in 96-well plates, the liquid bacterial growth inhibition test can be used to screen a large number of venoms. However, some venoms, inactive at 10 mg/mL in liquid media, have been found to be active, in the literature, by the solid media diffusion method. A comparative work between solid and liquid media was carried out using venoms described in the literature for their antibacterial activity. In liquid medium, venoms were tested at 10 mg/mL, and in solid medium at 100 µg.

### 3.1.2. Screening of venoms using Radial Diffusion Assay (RDA)

The RDA method facilitated the screening of the antimicrobial activity of 185 venoms at a final concentration of 100 µg against *Staphylococcus aureus* ATCC 6538P and *Pseudomonas aeruginosa* ATCC 9027
and to establish a comparative research between liquid and solid methods. The obtained results demonstrated that among the tested venoms, 113 samples were active against *Staphylococcus aureus* ATCC 6538P compared to 72 samples against *Pseudomonas aeruginosa* ATCC 9027. Furthermore, 82 samples were active against both strains.

## 3.2. Comparison of both methods

The comparison of the results revealed high degree of similarity in the activity of venoms using the two tested methods. However, five venoms are active only on solid medium *Agelena labyrinthica*, *Notechis scutatus*, *Opistophthalmus carinatus*, *Opistoththalmus glabrifrons*, *Pandinus imperator* (Tables 2 and 3).

i) *Agelena labyrinthica* spider venom was inactive in liquid medium against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027 but showed activity against both strains when tested is solid medium. *Benli and Yijit* (2008) demonstrated the antimicrobial activity of *Agelena labyrinthica* venom against Gram⁺ and Gram⁻ bacteria when tested in solid medium. This activity is attributed to its pore forming mode of action which depress the bacterial cell wall leading to shrinkage and loss of cytoplasm. (Benli and Yijit, 2008). Similar results were observed by *Nazife and Mehlika* (2008), who found the venom active against *B. subtilis*, *P. aeruginosa*, *Shigella*, *E. coli*, and *S. aureus* in solid medium (Nazife and Mehlika, 2008).

ii) *Notechis scutatus* snake venom was inactive in liquid medium against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027, with antimicrobial activity only against *S. aureus* ATCC 6538P in solid medium. Our result was confirmed by *Stiles et al.* (1991) who described the antimicrobial activity of *Notechis scutatus* venom using disc method at a final concentration of 188 µg/disc (Stiles et al., 1991).

iii) *Opistophthalmus carinatus* scorpion venom was inactive in liquid medium against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027.

<caption>**Table 2**</caption>

Comparison of the antimicrobial activity of venoms using two methods. Experiments were performed using LB media (*S. aureus* ATCC 6538P, *P. aeruginosa* ATCC 9027).

<table><thead><tr><th rowspan="2">Venom concentration<br>From 10 mg/ml Solution</th><th colspan="2">Microbroth dilution assay</th><th colspan="2">RDA</th></tr><tr><th colspan="2">400 µg</th><th colspan="2">100 µg</th></tr><tr><th>Venoms</th><th>S. aureus</th><th>P. aeruginosa</th><th>S. aureus</th><th>P. aeruginosa</th></tr></thead><tbody><tr><td>Bothrops jararaca</td><td>+</td><td>-</td><td>8</td><td>8</td></tr><tr><td>Buthacus arenicola</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Hadogene trogodytes</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Hottentata judaicus</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Opistophthalmus lawrencei</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Opistophthalmus<br>wahlbergii Tosca</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Opistophthalmus<br>wahlbergii Messina</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Parabuthus transvaalicus</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Hottentata franzwernerii</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Opisthacanthusasper</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Acanthophis praelongus</td><td>+</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Agelena labyrinthica</td><td>-</td><td>-</td><td>6</td><td>7</td></tr><tr><td>Bitis arietans</td><td>+</td><td>+</td><td>16</td><td>6</td></tr><tr><td>Bitis sgabonica gabonica</td><td>+</td><td>+</td><td>15</td><td>6</td></tr><tr><td>Crotalus adamanteus</td><td>-</td><td>+</td><td>19</td><td>11</td></tr><tr><td>Crotalus atrox</td><td>-</td><td>+</td><td>11</td><td>9</td></tr><tr><td>Echis (carinatus) sochureki</td><td>-</td><td>+</td><td>21</td><td>8</td></tr><tr><td>Naja annulifera</td><td>-</td><td>+</td><td>14</td><td>6</td></tr><tr><td>Naja mossambica</td><td>-</td><td>+</td><td>8</td><td>6</td></tr><tr><td>Notechis scutatus</td><td>-</td><td>-</td><td>9</td><td>-</td></tr><tr><td>Opistophthalmus carinatus</td><td>-</td><td>-</td><td>-</td><td>8</td></tr><tr><td>Opistophthalmus<br>glabrifrons</td><td>-</td><td>-</td><td>-</td><td>7</td></tr><tr><td>Pandinus imperator</td><td>-</td><td>-</td><td>8</td><td>6</td></tr></tbody></table>

9027, however it displayed antimicrobial activity against *P. aeruginosa* ATCC 9027 in solid medium. Through microbroth dilution method. *Moerman et al.* (2002), identified two amphipathic α helical peptides, opistoporin 1 and 2, from the venom which were active against Gram⁺ and Gram⁻ bacteria (Moerman et al., 2002).

iv) *Opistophthalmus glabrifrons* scorpion venom was only active in solid medium against *P. aeruginosa*. *Bao et al.* (2015) purified a cationic, amphipathic α helical opisin with 19 amino acid residues and without disulfide bridges from the venom. The antimicrobial activity of opisin was characterized using microbroth dilution method and it showed much higher activity against Gram⁺ bacteria than Gram⁻ bacteria (Bao et al., 2015).

v) *Pandinus imperator* scorpion venom exhibited antimicrobial activity against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027 when tested only in solid medium. Using disc method *Corzo et al.* (2001) tested the activity of two identified α helical polycationic peptides from *Pandinus imperator* venom which showed higher antimicrobial activity against Gram⁺ bacteria than Gram⁻ bacteria. The AMPs were named pandinin 1 and 2 and illustrated (Table 3) a primary structure of cationic amphipathic peptide with alternating cationic and hydrophobic residues and pore forming activity (Corzo et al., 2001).

The results analysis also demonstrated that *Acanthophis praelongus* venom was only active against *S. aureus* ATCC 6538P when tested in liquid medium at a final concentration of 10 mg/ml, with no activity observed against *P. aeruginosa* ATCC 9027 in both liquid and solid conditions. Our result was confirmed by *Francisc et al.* (2019) who demonstrated that *A. praelongus* venom was active against *S. aureus*; Methicillin-resistant *S. aureus* and *K. pneumoniae* with MICs of 2000, 4000 and 4000 µg/ml respectively and no activity was observed against *P. aeruginosa* and *E. coli* (Francisc et al., 2019). However, our result differed from those obtained by Perumal Samy (2007) who used the disc method to reveal an antimicrobial activity of *A. praelongus* venom against *S. aureus*, *P. aeruginosa*, *Proteus vulgaris* and *Proteus mirabilis* at a final concentration of 100 µg/ml (Perumal Samy et al., 2007). This difference may be due to the used media for antimicrobial activity screening; our screening was performed using LB, whereas Perumal Samy utilized MH media, which includes casein hydrolysate. This inclusion suggests that the active molecule may act synergistically with casein hydrolysate to enhance antimicrobial activity. Casein hydrolysates are known for their antimicrobial activity (Ouertani et al., 2018; Qi et al., 2023; Zhao et al., 2020).

*Naja annulifera* snake venom shows antimicrobial activity against *S. aureus* ATCC 6538P when tested on solid medium and against *P. aeruginosa* ATCC 9027 on both liquid and solid medium. *Blaylock* (2000) have demonstrated that *Naja annulifera* venom was active against *S. aureus*, *P. aeruginosa* and *E. coli* when tested on solid medium (Blaylock, 2000).

*Bothrops jararaca* viper venom demonstrated high antimicrobial activity against *S. aureus* ATCC 6538P in both liquid and solid medium. Additionally, it was active against *P. aeruginosa* ATCC 9027 only when tested on solid medium. *B. jararaca* venom inhibits promastigote forms of *Leishmania amazonensis* and epimastigote forms of *Trypanosoma cruzi*, causing rupture of the membrane and mitochondrial disruption (Gonçalves et al., 2002; Ciscotto et al., 2009). Using disc method, Ferreira et al. (2011) showed that *B. jararaca* exhibited an antibacterial profile against some of the Gram-positive and Gram-negative bacteria; *S. aureus*, *E. faecalis*, *E. coli*, *S. morcencens*, *P. mirabilis*, *P. aeruginosa*, *E. calcoacetic*, *A. calcoacetic*, *K. pneumoniae* (Ferreira et al., 2011).

Two scorpion venoms from *Androctonus amoreuxi* (origins Egypt and Mali) were inactive when tested on both solid and liquid medium (10 mg/ml). Antimicrobial peptides (AamAP1 and AamAP2) isolated from the venom of the North African scorpion *Androctonus amoreuxi* were active against *S. aureus* and *E. coli* with MICs of 20 and 150 mM
<table><caption>**Table 3**
Identified antimicrobial peptides in various venoms and summary of their mechanisms of action.</caption><thead><tr><th>Peptide</th><th>Origin venom</th><th>Amino Acids</th><th>Structural Features</th><th>Mechanism of Action</th><th>Molecular Weight (kDa)</th><th>References</th></tr></thead><tbody><tr><td>sLAAO</td><td>Bothriechis schlegelii</td><td>498</td><td>α-helical Flavoenzyme with oxidase activity</td><td>Generates hydrogen peroxide through the oxidation of l-amino acids, causing oxidative stress in bacteria</td><td>~60</td><td>Smith et al. (2014).</td></tr><tr><td>LAAO</td><td>Bothrops atrox</td><td>520</td><td>α-helical Flavoenzyme with oxidase activity</td><td>Produces hydrogen peroxide, leading to oxidative damage and cell lysis in bacteria</td><td>57</td><td>Costa-Oliveira et al., 2019.</td></tr><tr><td>Batroxicidin</td><td>Bothrops atrox</td><td>34</td><td>Cationic, amphipathic, α-helical peptide</td><td>Disrupts bacterial cell membranes by forming pores, leading to cell lysis</td><td>4.258</td><td>Lee et al. (2015).</td></tr><tr><td>Opistoporin 1</td><td>Opistophthalmus carinatus</td><td>44</td><td>Amphipathic α-helical peptide</td><td>Binds to bacterial membranes, causing membrane destabilization and pore formation</td><td>4.848</td><td>Clark et al., 2009a.</td></tr><tr><td>Opistoporin 2</td><td>Opistophthalmus carinatus</td><td>44</td><td>Amphipathic α-helical peptide</td><td>Similar to Opistoporin 1, it disrupts bacterial membranes leading to cell death</td><td>4.848</td><td>Clark et al., 2009b.</td></tr><tr><td>Pandinin 1</td><td>Pandinus imperator</td><td>44</td><td>Cationic, polycationic peptide with two α-helical</td><td>Interacts with negatively charged bacterial membranes, inducing membrane permeabilization and cell death</td><td>4.8</td><td>Garcia et al. (2010a).</td></tr><tr><td>Pandinin 2</td><td>Pandinus imperator</td><td>24</td><td>α-helical Cationic, polycationic peptide</td><td>Enhances antimicrobial efficacy through synergistic interactions with Pandinin 1</td><td>2.61</td><td>Garcia et al. (2010b).</td></tr><tr><td>AamAP1</td><td>Androctonus amoreuxi</td><td>18</td><td>α-helical Cationic, amphipathic peptide</td><td>Disrupts bacterial membranes by forming pores, leading to cell lysis</td><td>1.93</td><td>Martin et al. (2015a).</td></tr><tr><td>AamAP2</td><td>Androctonus amoreuxi</td><td>18</td><td>α-helical Cationic, amphipathic peptide</td><td>Similar to AamAP1, it targets bacterial membranes, causing membrane permeabilization and lysis</td><td>1.88</td><td>Martin et al. (2015b).</td></tr><tr><td>Opisin</td><td>Opistophthalmus glabrifrons</td><td>19</td><td>α-helical Cationic, amphipathic α-helical peptide</td><td>Binds to and disrupts bacterial membranes, inducing pore formation and cell death</td><td>2.167</td><td>Smith et al. (2021).</td></tr></tbody></table>

Note: All peptides are characterized by their α-helical structures, contributing to their antimicrobial mechanisms.

respectively (Almaaytah et al., 2012). The difference between our work and Almaaytah et al. (2012) may be due to the low abundance which appears to limit their defensive functions when tested on low concentrations (Gao and Zhu, 2018).

A bibliographic research was carried out on venoms found inactive using microbroth dilution method. Those not described in the literature were tested using the RDA technique. This allowed the identification of five additional active venoms *Hadogenes zuluanus*, *Mesobuthus caucasicus*, *Mylabris quadripunctata*, *Nebo hierichonticus*, and *Opistophthalmus wahlbergii*, with antimicrobial activity against *S. aureus* ATCC 6538P. These findings confirm that various methods should be used for detecting antimicrobial activity, rather than limiting to just one technique. A better efficiency in screening and identifying new antimicrobial molecules may help to address the problem of bacterial resistance in both human and veterinary medicines (Miethke et al., 2021).

Result analysis reveals that *Bitis arietans* and *Walterinnesia aegyptia* venom are active only against pathogenic strains *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027. Al Asmari et al. (2015) described the antimicrobial activity of *Bitis arietans* venom against *S. aureus* and *S. aureus* (MRSA) with no activity observed against *E. faecalis*, *E. coli* and *P. aeruginosa* (Al Asmari et al., 2015). Additionally, Okumu et al. (2023) demonstrated that both Gram positive and Gram-negative bacteria were resistant to *B. arietans* venom as no inhibition was observed (Okumu et al., 2023). The current work is the first research that shed light on the antimicrobial properties of *Bitis arietans* venom against *S. aureus* ATCC 6538P.

This work allows for the first time the identification of antimicrobial activity against *B. subtilis* ATCC6633, *P. aeruginosa* ATCC 9027 and *S. aureus* ATCC 6538P in venoms of snakes *Naja nubiae* and *Bothrops lanceolatus*, and this activity was confirmed using microbroth dilution assay and RDA.

Despite the difference in results, the diameter of the inhibition zones of venoms (*Agelena labyrinthica*, *Notechis scutatus*, *Opistophthalmus carinatus*, *Opistophthalmus glabrifrons*, and *Pandinus imperator*) is small and does not exceed 9 mm. These results suggest that the AMPs are present in very low concentrations and may be diluted when tested in liquid medium leading to the loss of their activity (Table 3). Agar plates are often used to visualize interactions between molecule and bacteria due to the manifestation of visible phenotypes that identify whether venom possesses antimicrobial activity (Stubbendieck et al., 2016). In

liquid medium bacteria is more resistant due to advantage in metabolism and/or proliferation (Paquette et al., 2018). They produce peptidases and proteases as secondary metabolites that degrade AMPs. Linear peptides are the main target for those enzymes; as proteolytic sites are more exposed to cleavage (Guilhelmelli et al., 2013). Some secreted molecules can bind to and neutralize the microbicidal activity of AMPs. For example, SIC proteins produced by *S. pyogenes* M1 bind to several AMPs, blocking their antimicrobial activity in the early stages of infection (Frick et al., 2003; Fernie King et al., 2004; Pence et al., 2010). Also, bacterial growth leads to acidification of extracellular medium. The reduced pH can inhibit the activity of AMPs (AbouAlaiwa et al., 2014). Furthermore, biofilms are well known for their remarkable resistance to various chemical, physical, and biological antimicrobial agents and are one of the main causes of persistent infection. Alginate, a polysaccharide in the *P. Aeruginosa* biofilm matrix, plays a significant role in the resistance of *P. aeruginosa* to AMPs (Davarzani et al., 2021). Another mechanism of bacterial resistance to AMPs is change in bacterial cell surface that block AMP access to their targets by affecting their binding and/or penetration into the cells (Guilhelmelli et al., 2013). Finally, Table 3 summarizes the physicochemical characteristics of all the proteins referenced in this study. The structure-activity relationship (SAR) analysis of the cited antimicrobial proteins highlights the significance of structural motifs such as amphipathic α-helices and cationic residues in promoting membrane disruption and pore formation in bacterial cells.

In this work, we compared two methods, the microbroth dilution and agar diffusion assays, for the rapid screening of 185 venoms, which allowed for the identification of antimicrobial-active fractions. However, other methods, such as the MTT-based colorimetric method (Oh and Hong, 2022), could be utilized in the future. Unlike the methods used, its application to bacterial assays may necessitate preliminary optimizations. Future studies will incorporate the MTT-based method and compare it to the methods utilized to validate and complement the antimicrobial activity assessments, thereby enhancing the robustness of our findings.

### 3.3. Antimicrobial molecule purification using high performance liquid chromatography HPLC

Several fractions were obtained by HPLC separation (Verdoni et al.,
2016; Chalier et al., 2020). After lyophilization, each fraction was analyzed for antimicrobial activity. These analyses revealed that:

In the case of *Bothrops lanceolatus* snake venom, fraction f8, which was eluted at 8 min retention time, demonstrated antimicrobial activity against both *S. aureus* ATCC 6538P and *P. aeruginosa* and no activity was described in the literature (Fig. 3A). For *Walterinnesia aegyptia* snake venom, fraction f13, with a retention time range of 39–41 min, exhibited antimicrobial activity against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027 (Fig. 3B). Concerning *Gloydius blomhoffii brevicaudus* snake venom, fractions f8 and f9, eluted at 8 and 9 min retention time respectively, displayed antimicrobial activity against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027 (Fig. 3C).

Our results on the venom of *Cheloctonus jonesii* reveal that two fractions, F2.0.3 and F2.3, demonstrate significant antimicrobial activity against *S. aureus* ATCC 6538P and *P. aeruginosa* ATCC 9027 (Fig. 4A). Furthermore, fraction F2.0.3 exhibited additional complexity with four distinct elution peaks, suggesting the presence of multiple active peptides (Fig. 4). These observations are corroborated by a recent study investigating the venoms of three buthid scorpion species from Colombia—*C. margaritatus*, *T. pachyurus*, and *T. n. sp. aff. Metuendus* (Mendoza-Tobar et al., 2024). This study identified several fractions with antimicrobial activity against the same bacterial strains. Specifically, *C. margaritatus* possessed two antimicrobial fractions, *T. pachyurus* had three fractions active against *S. aureus* and two against both *S. aureus* and *P. aeruginosa*, while *T. n. sp. aff. Metuendus* presented one fraction active against *S. aureus* and five fractions active against both bacterial strains. Additionally, some peptide fractions from these venoms were found to be toxic to mice, highlighting a delicate balance between antimicrobial efficacy and toxicity.

Comparing our results with those of the Colombian study highlights the diversity and specificity of antimicrobial peptides present in the venoms of different scorpion species. This comparison underscores the potential of scorpion venoms as a rich source of novel antimicrobial agents and emphasizes the importance of balancing therapeutic efficacy

with safety. In summary, integrating advanced fractionation methods such as Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) with systematic evaluations of antimicrobial activity and toxicity is essential for fully exploiting the therapeutic potential of animal venoms. Future research should focus on the structural characterization of the identified active peptides, the production of synthetic analogues by chemical means and the optimization of their antimicrobial properties while minimizing toxicity, paving the way for the development of innovative and safe antimicrobial treatments derived from venoms.

## 4. Conclusion

This research sheds light on the antimicrobial activity of various venoms sourced from animals. The results reveal that snake venoms, in particular, exhibit significant activity against a range of pathogenic bacterial strains. It's noteworthy that venoms from vipers, especially those from the Viperidae family (Crotalinae and Viperinae), proved more effective than those from the Elapidae family.

Tests showed that most venoms were active against *B. subtilis* ATCC 6633, *E. coli* ATCC 8739, *S. aureus* ATCC 6538P, and *P. aeruginosa* ATCC 9027, although some displayed selective activity against pathogenic strains. Additionally, the results underscore the importance of venom concentration in its antimicrobial efficacy, with some venoms losing activity at lower concentrations.

The testing methods employed, including liquid microdilution and solid diffusion testing, revealed variable yet complementary results. For example, some venoms showed activity only in one of the two media, highlighting the importance of selecting appropriate testing methods to characterize venoms' antimicrobial activity.

This research underscores the critical importance of employing high concentrations to detect the activity of peptides present in very low amounts and utilizing various screening methods to ensure the presence or absence of active peptides.

For the first time, we have identified antimicrobial activity in.

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Separation of *Bothrops lanceolatus* (A), *Walterinnesia aegyptia* (B), *Gloydius blomhoffii brevicaudus* (C) crude active venoms and their active fractions, F8 (A), F13 (B) and F8 & F9 (C).</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Separation of Cheloctonus jonesii venom and its active fraction F2.0 and F2.3 (A). Separation of active fraction F2.0 (B). Separation of active fraction F2.0.3 (C).</figcaption></figure>

* *Bitis arietans* venom against *P. aeruginosa* ATCC 9027.

* Naja nubiae and *Bothrops lanceolatus* venoms against *B. subtilis* ATCC 6633, *P. aeruginosa* ATCC 9027, and *S. aureus* ATCC 6538P.

* Venoms from scorpions such as *Hadogenes zuluanus*, *Mesobuthus caucasicus*, *Nebo hierichonticus*, *Opistophthalmuswahlbergii*, and *beetles like Mylabris quadripunctata* against *S. aureus* ATCC 6538P.

Furthermore, the work explored the possibility of purifying antimicrobial molecules from venoms through HPLC.

These findings highlight the potential of these venoms as sources of effective antimicrobial agents and contribute to the broader understanding of key factors critical for accurate detection of antimicrobial properties in animal venoms.

### CRediT authorship contribution statement

**Awatef Ouertani:** Writing – review & editing, Investigation. **Chloé Mollet:** Investigation, Writing – original draft, Writing – review & editing. **Yasmine Boughanmi:** Writing – review & editing. **Harold de Pomyers:** Resources. **Amor Mosbah:** Supervision. **Hadda-Imene Ouzari:** Resources. **Ameur Cherif:** Supervision. **Didier Gigmes:** Supervision. **Marc Maresca:** Supervision. **Kamel Mabrouk:** Conceptualization, Methodology, Validation, Writing – review & editing.

### Ethical statement

The authors hereby stated that all procedures involving animals were conducted in ethical manner. All experiments on animals were performed in the compliance with international principles of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes (Strasbourg, 1986).

### Funding

We thank the French Ministry of Higher Education, Research and Innovation (MESRI) and the ANRT for the CIFRE grant awarded to Dr. Chloe Mollet in collaboration with Latoxan. Special thanks go to KSAS Rémi and REYNES Jordy from Venom World, without whom this work would not have been possible

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

No data was used for the research described in the article.

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