An Innovative in Vivo Model for Bioassay-Guided Testing of Potential Antimicrobials

Abstract

The use of in vivo models is critical in determining clinical relevance of potential chemotherapeutic molecules. Albeit mammals are of physiological relevance, the use of nonmammalian animals to investigate therapeutic efficacy of potential molecules at an initial stage of clinical research can complement in vitro studies. Here we suggest the use of a simple and inexpensive in vivo locust model in exploring the efficacy of novel chemotherapeutic molecules and/or large chemical libraries using high-throughput experimentation without legislative restrictions.

Despite advances in therapeutic approaches, around 60 million people die every year due to the top 10 diseases, with cardiovascular, cancer, and infectious diseases as the leading killers.¹ Furthermore, outbreaks such as COVID-19 have resulted in renewed interest by pharmaceutical companies to generate new drug leads and/or disinfectants. The normal process to drug discovery involves bioassay-guided testing of large chemical libraries or a repertoire of target-specific molecules in vitro, followed by preclinical investigation of a handful of compounds using in vivo models. Although a plethora of in vitro assays are available at the initial screening stage, the availability of in vivo models is often restricted due to expense, the labor needed, and legislative adherence with ethical concerns. In this regard, in vivo vertebrate models are considered physiologically relevant, however in vivo invertebrate models at an early drug discovery phase can be advantageous in regards to ethical acceptance, expense, speed, and technical convenience and can result in meaningful n values. In particular, invertebrates such as locusts are easy to handle and are relatively large invertebrates. Up to 50 locusts can be retained in one cage, thus allowing the investigation of manifold experimental variables. Additionally, volumes of up to 20 μL can be injected in a single locust and their hemolymph, that is, blood, of up to 10 μL can be collected at various intervals of time without sacrificing the animal. Notably, studies have shown that human infections due to bacteria and parasites can be modeled in locusts,^2,3 suggesting that invertebrates such as locusts can be used as an in vivo model at an early stage of preclinical drug testing. Of note, there are significant analogies amid the innate immune responses of invertebrates and vertebrates, specifically as insects such as locusts have a blood-brain barrier (BBB) that is highly selective; and exhibits functional properties similar to that of vertebrates, demonstrating that insects can be a beneficial in vivo model.

Recently, locusts were utilized effectively to comprehend the invasion of pathogenic microbes into the central nervous system (CNS) in vivo.^2,3 Furthermore, inoculation of the protist pathogen Acanthamoeba, into the locust at the abdominal terga, when inserted at the intersegmental membrane in the hemocoel, caused locust mortality with more than 95% rate. To elucidate if the parasite associated with the CNS, the brains of the locusts were separated by eliminating the left side of the head, through an incision at the sagittal plane, and then dissecting the brain, utilizing forceps.² Next, utmost care was taken to ensure that both cerebral ganglia were in situ and free of any air sacs or fat body tissues, and all of the dissection equipment were sterilized with 70% ethanol, at each step. Histological inspection of the CNS revealed that parasites infected the locust CNS, and this is concomitant with the disturbance of the glial cell complex and perineurium cell, that forms the locust BBB. Following injection, amoebae that were viable were retrieved from locust hemolymph, and other tissues, including the muscle tissues and fat body, signifying that the amoebae caused parasitaemia, and had survived the innate immune system’s defenses, having spread hematogenously to further tissues and entering the CNS, an outcome that is harmonious with amoebic encephalitis in humans.

To demonstrate the effectiveness of antimicrobial molecules, and determine the usefulness of the innovative locust model, bacterial infection was induced in locust. Briefly, locusts were injected with 20 μL of Staphylococcus aureus (2 × 10⁶ c.f.u.) that resulted in 65% ± 6 mortality.⁴ To establish the efficacy of gentamicin in the novel locust model, insects were inoculated with S. aureus, followed up by the inoculation of gentamicin. Remarkably, gentamicin displayed potent efficacy in shielding the locusts from mortality facilitated by S. aureus, at the above-mentioned concentrations. Indeed, mortality was documented at 2% ± 0.5 following 5 days. Moreover, the negative control group of locusts injected with PBS depicted mortality rates comparable to locusts injected with S. aureus plus gentamicin. These findings suggested clearly that the use of insects such as locusts can be of value to determine in vivo efficacy of potential antimicrobial molecules.

Overall, there are significant similarities at the cellular and physiological level in locusts and vertebrates. Furthermore, as the molecular constituents of the BBB in insects such as the model organism Drosophila are analogous with vertebrates, it is conceivable that there are resemblances in the methods by which microorganisms can invade and enter the CNS of locusts and vertebrates. Of note, the utilization of Drosophila as a model can be challenging technically (i.e., incapacity to inoculate large volume, difficulty in dissection of brains, fat bodies, and muscles, extraction of hemolymph). Thus, we propose the application of the innovative locust model for initial screening of bioactive molecules. This will provide benefical leads for the logical development and evaluation of chemotherapeutic strategies, and will support a shift away from entire reliance on vertebrate models.

Furthermore, the ethical apprehensions with regards to investigation on vertebrates are driving such research into nonmammalian models, and models such as the innovative locust model extend major advantages of lower cost and speed, infrastructure required, and legislative restrictions. These suggested invertebrate models are an appropriate response to the requests of governments and the general public to reduce and/or replace the use of animals in research, particularly of mammals, as well as having minimal technical challenges. Thus, the locust model is an ideal invertebrate model and should be utilized for preliminary identification and screening of antimicrobial drugs, consequently delivering novel compounds for the logical development and assessment of chemotherapeutic interventions, and the use of this model will allow a shift from entire reliance on vertebrate models.

The proposed locust model depicted here has the capacity to determine efficacy of potential disinfectants/chemical libraries against human diseases, allowing fast, cost-effective, high-throughput experimentation with limited ethical concerns. Pharmacokinetic profiles involving hemolymph concentration, involvement of the CNS, and pharmacodynamic profiles (susceptibility, concentration versus time-dependent effects) can be tested readily to determine drug efficacy. Nonetheless, as the pharmacokinetic profiles of bioactive molecules can differ between humans and invertebrates such as locusts, that is, the latter having an open circulatory system that will eradicate drugs swiftly, data should be analyzed with apprehension and related to relevant data attained from clinical studies. If such physiological disparities are realized and accounted for with caution, locusts can be developed for understanding pharmacokinetics in vertebrates. Nonetheless, if utilized as a basic screening model, these invertebrates will provide approximate indications of the potential of novel bioactive molecules in regards to efficacy, toxicity, and optimal routes of administration.

Author Contributions

N.A.K. envisaged the concept. R.S. reviewed relevant literature and both R.S. and N.A.K. formulated the initial draft of the manuscript. Both authors finalized the manuscript. R.S. and N.A.K. are scientists with a lifelong interest in the field of medical microbiology. Both authors are guarantors for the manuscript and contributed equally.

The authors declare no competing financial interest.