ANIMAL MODELS IN ANTIBIOTIC STUDIES AND INFECTIOUS DISEASES

Research can be defined as a planned and scientific study carried out to shed light on any subject, to find a solution to a problem, to reach certain concepts, theories or laws.
Research is a planned scientific study to develop or contribute to knowledge that can be generalized.
You all know the importance of research in bringing medicine to this point today. The history of animal experiments dates back to the fifth century BC. During these years, Alkamion's physiology studies, Abdera's dissection studies and Hippocrates' studies in Southern Italy were recorded in historical records. 2/3 of the researchers who won the Nobel Prize in Medicine Most of them received these awards as a result of animal experiments. Experimental research using animals has played an important role in the development of modern medical treatment and continues to be an essential method to determine new drugs and methods for the treatment of new diseases.

The basis of animal experiments in medical sciences is based on studies defined as vivisection, which can be explained as tests, experiments and training exercises that are harmful to living animals.
Animals and humans are biologically different species. The results obtained from animal experiments cannot be applied to humans.
As an alternative to animal experiments, tissue culture and computerized studies can be tried.
Animals also have rights.
No researcher enjoys using animals. However, many researchers know that animal experiments are necessary in the fight against many diseases.
Animals, and especially mammals, are very similar to humans. In the past, treatments for many diseases were first tested on animals and then began to be applied to humans. Today, animal experiments continue in the fight against cancer and AIDS.
Alternatives to animal experiments can support but cannot replace animal experiments.

Animals themselves also benefit from research. Treatment of many animal diseases has also become possible as a result of animal experiments.
British Scientists William Russell and Rex Burch introduced the 3R rule in the late 1950s.
Reduction: Reducing the number of animals to be used in the experiment
Refinement: Minimizing the animal's stress and pain
Replacement: Using other alternatives instead of animals
Try animals from one of the following breeding systems according to their characteristics. can be chosen:
Inbred: It is a form of production by mating offspring born from one parent. The aim of this is to reduce genetic variability by reducing genetic heterogeneity and increasing homogeneity. The most commonly used method is the mating of brothers and sisters or young parents and their offspring.
Outbred: It is the random mating of animals from the same stock.
Selective breeding: It is desired to obtain different characteristics by mating unrelated parents.

It is the mating of animals from two different inbred populations. Physiologically, they are more uniform than their parents. However, they cannot be used when genitic uniformity is desired.
Laboratory animals are classified according to their hygienic conditions.
Depending on the nature of the planned experiment, experimental animals can be selected according to their microbiological characteristics:
Axenic animals: They are animals completely free of microorganisms. They are obtained by hysterectomy and grown with germ-free techniques in isolators.
Gnotobiotic Animal: They are like axenic animals, but A small number of nonpathogenic microorganisms may be present.
Specific Pathogen Free Animal:Animal purified from specific pathogenic factors
Monitored Animal:Animal housed with a low security barrier system. It is shown to be free of major pathogens by repeated checks.
Conventional Animal: Animal with unknown and uncontrolled microbial flora.

For the first time in the field of bacteriology, Robert Koch injected the pure culture of Anthrax into mice in 1876 and showed that Anthrax, which was epidemic in those years, was caused by bacteria. The rabbit was first used as a test animal in the diagnosis of bacilli by Delafond in Paris in 1856. The two cornerstones of antimicrobial chemotherapy are the treatment of infections in animals. The first is that Domagk showed in 1935 that sulfonamide was effective in pneumococcal infections in mice.
The second is the discovery of Penicillin. Fleming used Penicillin for culture isolation and only in some cases as an antiseptic. Until the 1940s, Florey and Chain demonstrated the powerful therapeutic activity of penicillin by using it first in mice and then in humans.
This led to two new breakthroughs.
1. Systemic bacterial infections can be treated with medications.
2. It paved the way for in vivo studies in antimicrobial drug research.

A new antibiotic created an infection model in laboratory animals and could not be applied to humans without proving its antimicrobial effect and toxicological safety. More than 1000 animal models have been developed for experimental chemotherapy since 1960.
The most commonly used method in the discovery of new antibiotics is in vivo infection models.
An ideal model should have the following features:
It should be as similar as possible to infections in humans.
Infection technique should be simple
Agent. organism
The site of entry must be the same or at least similar to that in humans
Spread in the body
Tissue involvement
The severity of the disease can be predicted
The course can be investigated
Duration
Response to chemotherapy Measurable
Repeatable
Rat
Mouse
Rabbit
Dog
Pig
Many experimental infection models have these features. Endocarditis, pneumonia in rats, urinary infection models in rats and pigs, osteomyelitis, meningitis etc.

Research using this and similar models has played an important role in elucidating the pathogenesis of many diseases. At the same time, the use and dosage of disease-specific antibiotics were again in line with the results obtained from these animal experiments.
Again, as a result of these models, answers to many questions were obtained:
When is a bacteriostatic drug as effective as a bactericidal drug?
When is the bacterial death rate important?
When is the bactericidal titration in serum important?
What is the optimal dose range?
Postantibiotic. What is the significance of the effect?
In short, animal models of infection are important in predicting the effectiveness and tolerability of antibiotics before using them on humans. However, it is also important in bringing new approaches to the treatment of infections.
In vitro studies provide important information about the effectiveness and spectrum of new antibiotics, but
Animal experiments provide the bridge between in vitro studies and clinical results.

Toxicity studies
Pharmacokinetic properties
Postantibiotic effect
Optimal dose and duration
United States Food and Drug Administration recommends that new chemotherapeutics be first investigated through animal experiments.
The Cruelty to Animals Act 1876 (UK)
Animal Welfare Act 1970. (USA)
European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (ETS 123) (March, 1986)
European Science Foundation
When creating experimental infection models, we remove animals from their natural environment and place them in an environment that causes stress and pain. The following questions may come to mind here:
Is it ethically right to manipulate animals?
Is it right to subject animals to painful and painful treatments for our benefit?
Considering both ethically and the economic size of in vivo studies, it would be appropriate to conduct in vitro studies first.
The following can be done within the scope of in vitro studies:

1. Determination of in vitro antimicrobial properties of substances
Determination of Minimal inhibitory concentration and Minimal Bactericidal Concentration against microorganism. When combinations are considered, knowing the potential interactions (interference or antagonism, additivity, or synergism are learned through in vitro studies. Knowing the MIC and MBC are necessary issues before starting in vivo studies. For an antibiotic that will be effective under in vivo conditions (pH, pO2, pCO2), similar conditions must be created in vitro. Because environmental conditions significantly affect the antibiotic activity. However, microorganisms grow more slowly in in vivo conditions. They grow. This may affect the sensitivity of antimicrobial agents.
2. In vitro toxicity determination
These studies are generally performed in cell cultures, but they are not as safe as in vivo. In toxicity studies, the concentration of the antimicrobial drug must be above the MIC and MBC.

3. Knowing the solubility and stability of the antimicrobial agent in solution and preparing the most appropriate formula to be applied to animals
Knowing the solubility of the agent to be applied guides us about the route of administration and its pharmacological activity. In general, the substance should be prepared in the most soluble form.
When oral administration is considered, it should be known that there is a difference between species in absorption from the gastrointestinal tract.
For maximal bioavailability, the best formula should be prepared and the study should be carried out in the appropriate animal. The age of the animal is also important.
Knowing the degree of serum protein binding
The pharmacokinetics of free antibiotics that do not bind to serum proteins are the predictors of the best antibiotic activity for humans. Because free antibiotics are best at penetrating tissue fluids.

In vivo tests help us determine the advantages and disadvantages of new antibiotics in terms of activity, pharmacokinetic profile, route of administration and potential toxicity problems. Beta-lactamase inactivation observed in vitro may not occur in vivo. In many in vitro situations, very low MIC values may not reflect in vivo values. For these reasons, the known In vivo testing may be required for new compounds or derivatives of antibiotics.
Relationships between in vivo and in vitro results
Many substances that are active in vivo are also active in vitro, but the reverse is not always true.
It has the most significant effect on the in vivo activity of antibiotics. As the inoculum size increases, it results in a decrease in antibiotic effectiveness.

In animal experiments, the preparation of the infectious inoculum significantly affects the results. Essentially, the microorganism must be at maximum viability and appropriate culture media must be used when preparing the inoculum. The culture medium also affects the virulence of microorganisms.
One of the most important problems in animal experiments is the changes in the virulence and reproduction rates of microorganisms even though the same standard experiments are performed.
Generation time (bacterial cell division rate) may differ from in vitro. In vivo generation time increases progressively during infection and can take up to 20 hours depending on the site of infection. Prolonged generation time adversely affects the activity of antimicrobial agents.
The virulence of some microorganisms may be too low for a non-natural animal host, so some adjuvants may need to be added to establish infection.
Animal models most frequently used in the evaluation of antibiotics
Basic screening
Ex-vivo

Discriminative
Basic screening tests are generally used in the evaluation of new antibiotics.
Ex-vivo dosing schedule is used to determine specific variables such as serum binding or penetration into the extravascular space.
Discriminative systems are used to distinguish new agents from related or unrelated active agents.
Basic screening models
one-stage infection,
simple techniques and treatment applications,
short-term experiments,
repeatable,
easy to evaluate, low-cost tests.
The best example of these tests is the Mouse protection test.
It is the most frequently used in vivo research in antibacterial research. Mouse protection testing is the most appropriate method to investigate the effectiveness and toxicity of new antibacterials. It also gives information about whether the drug will be active orally or parenterally.

Different types of mice can be used in the mouse protection test for the reasons stated below.
It establishes a good relationship between the clinical response and activity to the antimicrobial agent.
A large number of animals can be present.
It is economical because the usage rate of the tested compound is low.
It is a heterogeneous population because outbred animals are used. This ensures immunological and other host differences.
It is necessary to use human pathogens whenever possible in developing animal experiments for in vivo testing. The microorganism used must be sufficiently virulent so that there is no need for additional applications to reduce the host's resistance. If reproducible infection cannot be achieved by inoculating the microorganism alone, it is necessary to apply adjuvant stressful procedures to reduce the host's resistance.
Using IM corticosteroids before inoculating the microorganism
Using cyclophosphamide
Amount of the drug
The most appropriate route of administration

To determine these, some toxicity studies can be performed in vitro, if possible. These include
a single injection of the drug orally, subcutaneously or parenterally in animals grouped in 6 conditions giving and then giving the animals It is based on observation between 24 hours and 7 days. Such a toxicity study would include either the 100% toxic dose (LD 100) or the 50% lethal dose (LD 50) of each route. and determines the Maximum Tolerable Dose (LD0) experienced by all animals. An estimated one-fifth of the maximum tolerated dose can be given as a single injection per day as a therapeutic dose. Oral bioavailability can also be calculated.
Determination of inoculum for infection: Determination of virulence

In order to create reproducible infections, it is necessary to determine the virulence of each strain. Appropriate cultures are taken for virulence tests. These cultures are diluted until the number of microorganisms is reduced by 10 times. 0.5 ml of each dilution is given intraperitoneally to 6 groups of rats. Animals are observed for 3-7 days. The number of live animals in each dilution is recorded. All animals are dead The lowest dilution is determined as the Minimal Lethal Dose (MLD). The dilution of MLD between 100 and 1000 is used for chemotherapeutic studies.
Establishing infection:
0.5 ml of the appropriate dilution solution of the infecting inoculum is given intraperitoneally.
Diluted solutions of the drug prepared in water It is given via gavage in 0.5 to 1 ml. If it is known that the drug is not absorbed from the gastrointestinal tract, the subcutaneous route is preferred.

Animals are observed over a 7-day period after being infected and treated. This period may vary between 3 and 14 days. The number of animals surviving during this period is recorded. A 50% protective dose (PD 50) is calculated. PD50 Same as 50% curative dose or 50% effective dose.
From all animals that died A blood sample is taken from half of the living people and planted on agar. Culture of dead animals should show infecting microorganisms, while culture of living animals should be sterile. If the culture of deceased animals is sterile, the possibility of drug toxicity should be investigated.
The subcutaneous route is more effective than the oral route.
Administration of chemotherapeutics immediately after infection occurs or 1 hour later is more effective in terms of protecting the animals.
Multiple applications may be required to determine activity.
Mouse practice in the test. Treatment is usually effective immediately after infection occurs.

In prophylaxis applications, treatment must be applied along with the infection.
The biggest difficulty of in vivo animal experiments is how effective the results are for humans. In most discriminative animal experiments, the experimental results are clinically similar.
Nonfatal subcutaneous infection method to determine topical antibacterial activity
Many antibiotics that are not active in systemic infections can become active when applied to the infection site.

There are many techniques to create local infection. Swiss albino mice are infected subcutaneously with 0.2 to 0.5 ml of a 24-hour culture of the microorganism. The animals are then injected into the infection site with 1ml of varying doses of the antibacterial agent. At the end of 24 hours, the animals are killed and a swab is taken from the infection area and planted in the appropriate agar medium. The absence of any growth or growth of more than 10 colonies is an indication of successful treatment. In these experiments, it is necessary to use a control group in which an antibiotic known to be effective against the causative microorganism is given.
The Selbie method is a sensitive infection method in determining the effectiveness of the antimicrobial agent, determining drug-pathogen interactions, and determining drug pharmacokinetics in the infected host. If the animals are made neutropenic, the Selbie method is safer in evaluating drug-microorganism interactions. This method is widely used to determine the pharmacokinetics of antibiotics and to determine the postantibiotic effect.

The method used by Vogelman et al. is a good example of this.
23-27 g Swiss outbrad mouse was used. The mice were made neutropenic by administering cyclophosphamide (100-150 mg intraperitoneally) on days 0 and 3. On day 4, severe neutropenia (less than 100 neutrophils per microliter) was observed. This situation continues for 2-4 days. The infecting microorganism is grown in culture and 0.1 ml is injected into both thighs on the 4th day. Infection is expected to develop over a 2-hour period. Then, varying doses of antimicrobial agent are administered subcutaneously to infected mice in groups. Infected but untreated animals are used as controls. 2-4 mice from each group are killed every hour within the first 4 hours. During the next 16-hour period, they are killed every 2 to 4 hours. In each sample, thigh muscle is taken and homogenized (in 9ml 0.85% NaCl) and after appropriate dilutions, log CFU/thigh is calculated. This method is widely used for PA effects and other interactions.

0.5 ml of the solution containing 1x109 to 2x109 staphylococci is inoculated intraperitoneally into female Swiss mice. Starting 3 hours after inoculation and at regular intervals thereafter, the animals are killed and abscess foci or parts where microorganisms accumulate are aseptically removed from the peritoneal cavity and then the bacterial population is calculated.
S. It is given intranasally from the culture containing pneumonia. Infected animals are killed after 48 hours, their lungs are removed aseptically, and lung tissue is cultured in blood agar. Alternatively, lung tissues are homogenized and homogenates are produced in culture.
Muse and rats are the animals most commonly used in the pathogenesis of urinary tract infections and chemotherapy.
Ascending obstructive pyelonephritis
Direct inoculation is made into both poles of the kidney. Antibiotherapy is started 24 hours after inoculation.

This model is the most commonly used method to create experimental UTI. Urethra is clamped. A 1cm midline incision is made just above the penis. The bladder is explored. Urine in the bladder is aspirated and bacteria is inoculated into the bladder. Urethral clamping can be continued for a while.
Transurethral inoculation
It is an ascending UTI model, but excessive inoculation should not be done to cause reflux.
Peritonitis can be caused either by direct intraperitoneal inoculation of fecal material or by cecum ligation and It is created by the perforation method.

In the rat model that mimics intra-abdominal sepsis, either a known microorganism or fecal culture is used. In both methods, the inoculum prepared in gelatin capsules is placed into the abdomen through a midline incision. Animals are killed on the following days 1, 3, 7 and 14. In the control group, sterile gelatin capsules are used. The killing process is also done under sterile conditions. The abdomen is shaved, cleaned with povidone-iodine, and either a peritoneal fluid sample or an aerobic or anaerobic culture is taken from the obvious abscess focus. Blood cultures are also taken. Blood cultures usually show growth of E.coli. In peritoneal cultures, it is observed that E.coli and Bacteroides strains are dominant in the early stages, and in advanced stages and in cases where abscesses develop, B. Fragilis v Fusobacterium strains predominate and small amounts of E. Coli and enterococci are present.

Postsurgical sepsis model is the most suitable example to develop lethal peritonitis. Laparotomy is performed under anesthesia. The cecum along with a part of the large intestine It is removed through the incision, the fecal material in the large intestine is reduced into the cecum, and the intestine is ligated distally. The cecum is punctured in two places with a 20-gauge needle and a small amount of cecal material is removed from the lumen. The cecum is returned to the abdomen and the abdomen is closed. Normally 1ml of physiological saline is given subcutaneously to prevent hypotension. In this method, the mortality rate is observed to be 80% in the first 48 hours. Although it is not a suitable method for chemotherapy, it is a suitable model as a sepsis model.