In vivo and In vitro Model for Evaluation of Anti-microbial activity: A Review

 

Saema¹, Tabassum Wasim Ahmed², Peeyush Kumar Sharma¹, Imran Khan Pathan¹, Mamta Bhatia¹, Marhaba Khan³

¹Department of Pharmacognosy, Faculty of Pharmacy, Maulana Azad University,

Vill: Bujhawad, Teh: Luni, Jodhpur-342802, Rajasthan.

²Sant Gadge Baba Amravati University (SGBAU) Camp Area,

Near Tapovan Gate, Amravati, Maharashtra 444602, India.

³Department of Pharmaceutics, Faculty of Pharmacy, Maulana Azad University,

Vill: Bujhawad, Teh: Luni, Jodhpur-342802, Rajasthan.

 

ABSTRACT:

The rates of infectious diseases have significantly increased in recent years. So need for production, evaluation and standardization of new antimicrobial agents has also increased. The major problem of resistance towards antibacterial, antiviral, etc drugs is also posing a great threat to human beings as many bacteria, viruses and fungi have become resistant to commonly used drugs. However, microbes are also useful for us in our basic physiological functions, for example: digestion, so we also need to make sure the new molecules of antimicrobials do not cause harm to our natural microbial flora. In this review, we have tried to assemble some In-Vivo and In-vitro Model for evaluating Antimicrobial agents. Classifications of antibacterials, antivirals and antifungals have been displayed in an easy way. The use of herbal drugs is displayed in a tabular manner and finally the evaluation parameters have been discussed.

 

 

 

INTRODUCTION:

In past decades, need to research for novel antimicrobial agents is being increased to fight against the present drug resistant micro-organisms. So estimating and assessing the antibacterial, antiviral, antifungal, etc activity of drugs is increasingly becoming important.

 

Finding out the antimicrobial capability of drugs can be used to predict therapeutic outcome, epidemiology and even discovering new drugs. This review has centered vast range of assays to test the action of antimicrobial drugs in-vitro and in- vivo both.¹

 

Antibiotic resistance is causing concern for increased deaths from microorganisms by 2050. Staphylococcus, which is able to form biofilms, is a major concern. Biofilm formation is responsible for 65-80% of all human infections and makes antimicrobial treatment ineffective.

 

Biofilm development involves initial attachment followed by extracellular matrix production through quorum sensing, resulting in improved resistance to antimicrobial drugs.²

 

The usual methods for determining antibiotic activity, either alone or in combination, generally expose a growing bacterial inoculums to a constant or static concentration of one or more drugs for a period of approximately 20-24 hr. Such methods include disk susceptibility tests, determinations of minimal inhibitory and minimal bactericidal concentrations with micro titer or macro tube dilutions and time-kill studies. Even the determination of serum bactericidal activity in patients or volunteers receiving a given antibiotic regimen only ascertains the antibiotic activity at a specific time, at a single concentration of drug or drugs.³

 

Role of microbes in human body⁴:

The human body contains a vast number of microorganisms known as the human microbiome or microbiota, which include bacteria, viruses, fungi, and protozoa. These microbes play a crucial role in maintaining the healthy operation of the human body's immunological, metabolic, and physiologic systems, particularly in the gastrointestinal system. The composition of the microbiota is influenced by factors such as nutrition, medication, genetics, and lifestyle. While some microorganisms cause infectious diseases, others act as commensals that help protect the body against harmful microbes. Overall, microbes are essential to human health and disease.

 

Body defense mechanism for countering microbes⁵:

Pathogens must come into contact with a host and create an infection focus before causing illness. Host defense mechanisms, including the epithelial surfaces and tissue macrophages, prevent pathogen colonization. Inflammatory responses trigger innate immunity, which engages pathogens directly and resists future challenges. These defenses often stop infections from taking hold, but if not, triggered responses recruit and create more effector molecules and cells, aiding in an adaptive immune response.

 

Drugs used as antibacterial, antiviral and antifungal Agents:

Antibacterial agents (antibiotics)⁶:

Antibacterials are used to treat bacterial infections. The most common groups for antibacterials are beta-lactams, macrolides, quinolones, tetracyclines, and aminoglycosides. Their pharmacodynamics, chemical makeup, and antimicrobial spectra determine where they fall under these groups.

 

Table 1 Mechanism of action of Antibacterial drugs of different categories

 

Antiviral drugs⁷:

Viruses are major pathogenic agents causing a variety of serious diseases in humans, other animals, and plants. Antiviral medications are those that fight viral infections. For many viral infections, there are no efficient antiviral medications. However, there are a number of medications for treating influenza, a few for treating herpes, and a few novel antiviral medications for treating HIV and hepatitis C infections.

 

Table 2 Targets and mechanisms of action of Antiviral drugs respective to the viral agents.

 

Table 3 Mechanism of action of different classes of antifungal drugs

 

One of the most pervasive forms of life, viruses may infect every kind of animal, from mammals to insects, plants, and even microorganisms. The earth seems to have more virus species than all other animal species combined. Antiviral medications that directly target viruses include integrase inhibitors, protease inhibitors, un-coating inhibitors, nucleoside and nucleotide reverse transcriptase inhibitors, and attachment, entrance, and un-coating inhibitors.⁸

 

Antifungal agents:^9,10

Fungal infections have been on the rise since the 1950s, partly due to the use of broad-spectrum antibiotics, corticosteroids, anticancer/ immunosuppressive medications, dentures, indwelling catheters and implants, and the introduction of AIDS. Saprophytic fungi can easily invade live tissue due to weakened host defenses. (Table-3).

 

Topical antifungals and systemic medications like griseofulvin, amphotericin B, imidazoles, triazoles, terbinafine, and echinocandins have been developed to treat fungal infections. Echinocandins are the most recent addition to antifungal medications.

 

Mechanism of Antibacterial Action:

There are several mechanisms of antibacterial action, including:

·       Inhibition of cell wall synthesis: Antibiotics like penicillin, cephalosporins, and carbapenems work by inhibiting the synthesis of the bacterial cell wall, leading to cell lysis.

·       Inhibition of protein synthesis: Antibiotics like macrolides, tetracyclines, and aminoglycosides target bacterial ribosomes and prevent protein synthesis, leading to bacterial death.

·       Inhibition of nucleic acid synthesis: Antibiotics like quinolones and metronidazole inhibit bacterial DNA synthesis, leading to bacterial death.

·       Disruption of membrane function: Antibiotics like polymyxins interact with bacterial cell membranes, causing membrane disruption and cell death.

·       Inhibition of metabolic pathways: Antibiotics like sulfonamides and trimethoprim inhibit bacterial metabolic pathways, leading to bacterial death.

·       Inhibition of enzyme activity: Antibiotics like beta-lactams and sulbactam inhibit bacterial enzyme activity, leading to bacterial death.

 

IN-VIVO MODELS FOR EVALUATION OF ANTI MICROBIAL ACTIVITY:

Tissue-Cage Infection Model (Antibacterial activity evaluation):¹¹

This model is conducted in Calves. Two sterile golf practice wiffle balls (43mm in diameter, with a volume of 34 ml, as shown in Figure) will be implanted subcutaneously in calves.

 

Figure 1: - The tissue cages used in in vivo antimicrobial activity of marbofloxacin against Pasteurella multocida in calves.

 

After surgical intervention, the calves will be treated with antibiotic for 3-5 days, BD to prevent any infection. After 4 weeks of implantation, each tissue-cage will become sealed with a thin layer of connective tissue and filled with clear, yellowish tissue cage fluid (TCF). Afterwards, sterility of TCF sampling will be assessed using aerobic and anaerobic culture of sample from each cage and sampling of TCF will done using percutaneous puncture.

 

Approximately 5 x 10⁶ CFU of exponentially developing P. multocida culture will be suspended in 100 l of sterile isotonic saline and then injected into each pre-implanted wiffle ball. Following inoculation (-24h), tissue cage fluid will be collected at 0, 3, 6, and 24 hours after drug delivery. Using 0.9% NaCl, each sample (50 l) will be treated to 10-fold serial dilutions. On BTSA agar, 25 micro liters of each dilution will be plated in quadrants. The plates will then be incubated overnight at 37°C and vitality will be measured. The detection limit (LOD) should be 400CFU/mL.

 

Rabbit skin burn infection model:¹²

Rabbit of either sex will be infected with E. coli. The rear hair of the animals will be trimmed and then removed using a paste of barium sulfide-zinc oxide-starch (2:3:3, wt/wt/wt) one day before infection. On the day of infection, the rabbits will be sedated with an intravenous injection of pentobarbital sodium (30mg/kg of body weight), and their back skin will be sterilised with 75% ethanol. On the back of each animal, seven severe second-degree burn wounds (six for giving chemicals and one as a control) will be made by using a brass probe (1.8cm in diameter, 100°C) for 10 seconds. After 15 minutes, 0.1ml (challenge dosage, 1 x 10⁷ CFU/burn wound) of an E. coli suspension in saline will be subcutaneously injected into each wound, and a handmade cap will be applied to each lesion for protection. One hour after the burn, 0.4ml of various compound solutions or saline (for the control) will be loaded onto a sterile gauze patch of the same size as the burn site and applied to the respective wounds. To keep the wounds moist, sterile petrolatum-containing gauze should be applied over the gauze holding the compound. The rabbits will be slaughtered 24hours after the chemicals are administered. Then, sterile full-thickness skin biopsies will be obtained from the centre of the burns and homogenised in saline. The homogenates will be serially diluted 10-fold, and 0.1-ml aliquots will be plated on nutritional agar. The plates will be incubated at 35°C for 48hours, and the number of live organisms (CFU per gram) in the burn sites will be measured.

 

Mouse ascending urinary tract infection model:¹²

Rodents will be utilised to investigate the therapeutic efficacy of a medication against ascending urinary tract infections caused by E. coli. Prior to and after infection, the animal will be placed on a 24-hour water restriction. Under pentobarbital anaesthesia, a round-point needle will be inserted transurethrally to inject 0.05ml of the bacterial solution in 5% mucin (2.2 x 10⁹ CFU/animal challenge dosage) into the bladder. After inoculation, the urethral needle will be withdrawn and the external urethral meatus will be constricted for one hour. The compounds (saline for the control group) will be delivered subcutaneously six, twenty-four, thirty, forty-eight, and fifty-four hours after infection. 72hours following infection, animal will be sacrificed. After removing and homogenising the kidneys in saline, 0.1-ml aliquots will be placed on nutrient agar plates. After 48hours of incubation at 35°C, the number of viable organisms (CFU per gram) in the kidneys will be calculated.

 

IN-VITRO MODELS FOR EVALUATION OF ANTI MICROBIAL ACTIVITY:

Agar disk diffusion method:¹

Numerous clinical microbiology labs utilise the Agar disc-diffusion test to conduct standard antibiotic susceptibility testing. Although not all fastidious bacteria can be accurately tested with this method, it has been standardised to test disease-causing bacteria such as streptococci, Haemophilus influenzae, Haemophilus parainfluenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, using specific culture media, different incubation conditions, and interpretive criteria for inhibition zones. In this approach, a standardised inoculum of the test microorganism is used to inoculate agar plates. Then, filter paper discs containing the test substance at the specified concentration are deposited on the agar surface. Under favourable circumstances, the Petri dishes are incubated. Typically, an antimicrobial agent diffuses into the agar and inhibits the germination and development of the test microorganism, followed by the measurement of the diameters of the growth inhibition zones. Antibiogram yields superior outcomes by classifying bacteria as vulnerable, intermediate, or resistant. This approach cannot discriminate between bactericidal and bacteriostatic effects, since the suppression of bacterial growth is not synonymous with bacterial death. In addition, the agar disk-diffusion technique is unsuitable for determining the minimum inhibitory concentration (MIC) since it is hard to measure the quantity of antimicrobial agent that diffused into the agar medium. In spite of this, an approximation of the MIC may be determined for some microbes and drugs by comparing the inhibitory zones with stored algorithms. However, disk-diffusion assay provides several benefits over other methods: Simplicity, cheap cost, the capacity to test a vast array of microorganisms and antimicrobial agents, and the ease of interpreting data.

 

Inhibition of virus induced cytopathic effect (CPE)¹³

A quantal test may be used to assess the efficacy of an antiviral drug against viruses that produce CPE but do not easily form plaques in cell cultures. The test will be conducted as follows: a series of quadruplicate cell cultures will be infected with a continuous dosage of around 100 TCIDs0 of virus. After 1-2 hours of virus adsorption at 37°C, different antiviral maintenance medium doses are administered to the cultures. If feasible, the concentration range should encompass a low dosage at which no antiviral activity is seen and a high dose at which virus replication is maximally suppressed. Each day, virus-induced CPEs are documented until all quadruplicate cultures in the virus control (i.e., cultures without the antiviral drug) exhibit CPE. The 50% effective dosage (EDs0) of the antiviral drug is the concentration required to stop CPE in half of the quadruplicate cultures. Alternately, the degree of CPE caused by the virus may be compared to various concentrations of the antiviral agent, and the EDs0 will be the drug concentration that reduces CPE by 50%. A viable cell count test for the measurement of anti-HIV activity in cultured cells has been developed. Protection of viable cells demonstrates the antiviral action of an antiviral drug against HIV infection, given that HIV infection reduces the number of viable cells in infected cultures.

 

Plaque reduction assay¹³:

Plaque reduction assay is a straightforward and efficient method for assessing antiviral efficacy against viruses that produce plaques in compatible cell systems. Typically, this is carried out in cell monolayers infected with a steady dosage of the virus, such as 50-100 PFU (plaque forming units), depending on the size of the cell monolayer. After 1-2 hours of viral adsorption at 37°C, infected cell monolayers will receive an overlay medium with nutrients and 1-2% methylcellulose containing a variety of test agent doses. The infected cultures are then reintroduced to incubation for a length of time that varies depending on the virus. For counting plaques, infected cultures are preserved and stained for observation. Herpes simplex virus type 1 (HSV-1) plaque formation was inhibited by acyclovir in Vero and guinea pig embryo (GPE) cell monolayers, as illustrated in Figure 1. (ACV). By comparing the plaque number obtained in virus-infected cultures without the drug (controls) to the plaque number obtained in virus-infected cultures containing varying concentrations of the drug, a dose response curve will be obtained, and the dose required to reduce the plaque number by 50% (ED50) will be determined.

 

EVALUATION PARAMETERS:

Time-kill test (time-kill curve)¹:

Time-kill test is the most suitable approach for assessing bactericidal or fungicidal action. It is an effective instrument for acquiring information on the dynamic interaction between the antimicrobial agent and the microbiological strain. The time-kill test demonstrates an antibacterial action that is time- or concentration-dependent. Three tubes holding 5*105 CFU/mL of a bacterial suspension will be used to conduct the experiment in broth culture medium. The first and second tubes will typically contain the tested molecule or extract at final concentrations of 0.25* MIC and 1* MIC, respectively, while the third tube will serve as a growth control. The incubation will be conducted under optimal circumstances across a range of times (0, 4, 6, 8, 10, 12 and 24h). The proportion of dead cells will then be determined compared to the growth control by calculating the number of live cells (CFU/mL) in each tube using the agar plate count technique. In general, a bactericidal effect may be achieved with a lethality percentage of 90% for 6h, which is comparable to 99.9% for 24h. In addition, this approach may be used to assess synergism or antagonism between medications (two or more) in combination. Using the same methodology, a number of antifungal compounds have been investigated.

 

ATP bioluminescence assay:¹

The ATP bioluminescence test is used to quantify the concentration of ATP generated by live cells and assess microbial population inside a sample. This test works by converting D-luciferin to light-generating oxyluciferin using luciferase in the presence of ATP, and the amount of light emitted is measured using a luminometer. The bioluminescence assay has many applications, including cytotoxicity tests, assessment of biofilm influence, and drug screening against microorganisms. This method is quick and can produce antimicrobial test results in 3-5 days compared to the standard dilution procedure that takes 3-4 weeks of incubation.

 

Flow cytofluorometric method¹⁴:

Flow cytofluorimetry is an objective and precise method to evaluate the number and level of expression of surface markers on cells. It is also useful for studying per-cell cell death and apoptosis, where changes in the expression of surface proteins can be evaluated using a fluorescent substrate tagged with an antibody. Cytofluorometric methods are becoming more popular due to their simplicity and ability to provide quantitative data quickly. The simplest technique involves labeling cells with a chromatinophilic dye and comparing apoptotic cells to cells in the main peak. Alternatively, annexin V can be used to identify exposure of PS on the plasma membrane. Several modern cytofluorometric assays have been developed based on different functional features of cell-permeant probes.

 

CONCLUSION:

Infections caused by microorganisms have become a substantial clinical danger, with considerable morbidity and death, mostly owing to the evolution of microbial resistance to conventional antibiotic drugs. Consequently, technologies for antimicrobial susceptibility testing and the discovery of new antimicrobial drugs have been widely used and continue to be developed. Antimicrobial susceptibility testing against pathogenic microorganisms is the laboratory's most important responsibility. The testing's primary objective is to determine the frequency of pathogenic medication resistance and the certainty of susceptibility to certain infection medicines. This information aids in the selection of antimicrobial agents, the creation of antimicrobial policies, and offers observational data. Therefore, the purpose of this paper is to examine the in vitro and in vivo procedures of antimicrobial bioassays of diverse drugs against a variety of harmful bacteria.

 

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

 

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Received on 02.01.2023         Modified on 07.02.2023

Accepted on 13.03.2023   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2023; 13(3):169-174.