Antibiotic susceptibility testing: preparation, deciphering, how much is done

Today, antibiotic sensitivity testing is becoming increasingly popular. Human microflora is quite diverse, represented by a large number of microorganisms in various biotopes.

Pharmaceutical companies have developed a large number of antibacterial agents, antibiotics, which help maintain a normal ratio and number of microbial populations. With the onset of the antibiotic era, many diseases that were previously considered fatal have been cured. But microorganisms also strive to survive, gradually adapting to the action of antibacterial drugs. Over time, many of them have acquired resistance to many drugs, fixed it in the genotype and began to pass it on from generation to generation. Thus, new microorganisms are initially insensitive to certain drugs, and their use may be ineffective. Pharmacists are developing more and more new products, adding new active components to them, changing the basic formula. But gradually, resistance to them also develops.

The reason for the increased resistance of microflora to many drugs, and even their analogues, is often hidden in the incorrect and uncontrolled use of antibiotics. Doctors prescribe antibiotics and their combinations for various bacterial diseases. At the same time, there is no preliminary assessment of how effective they will be, the optimal dosage is not selected, which is very important both for treatment and for preventing the mechanisms of development of further resistance. Many people mistakenly prescribe antibacterial therapy even for viral diseases, which is ineffective, since the antibiotic does not act against viruses.

Therapy is often prescribed without preliminary sensitivity testing, the selection of the active agent and the required dosage for each specific disease and biotope is not performed. Since antibiotics are prescribed "blindly", there are often cases when they do not show any activity against the microorganisms that caused the disease and whose numbers need to be reduced. Instead, they affect other representatives of the microflora, resulting in dysbacteriosis, which is also a rather dangerous pathology and can lead to serious consequences. Particularly dangerous are cases when an antibiotic destroys the normal microflora, which is designed to protect the body and maintain its normal functioning. There are also cases when too much or too little of the drug is prescribed.

Patients are also irresponsible about treatment. Often, treatment is stopped after the symptoms of the disease have ceased to bother. At the same time, many prefer not to complete the full course. This is one of the factors that contribute to the development of resistance in bacteria. The full course is designed to completely kill pathogenic microflora. If the course is not completed, it is not completely killed. Those microorganisms that survive undergo mutations, develop mechanisms that provide them with protection from this drug, and pass it on to the next generations. The danger is that resistance is developed not only in relation to this specific drug, but also to the entire group of drugs.

Therefore, today one of the most effective means of rational therapy and prevention of resistance is the preliminary determination of sensitivity to the prescribed drug and the selection of its optimal dosage.

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Indications for the procedure antibiotic sensitivity testing

Normally, such an analysis should be carried out in all cases where antibacterial therapy is required. Based on the basic laws of antibiotic therapy, any antibiotic can be prescribed only after a preliminary assessment of the sensitivity of the microflora to this agent has been carried out, and the optimal concentration of the active substance has been determined in laboratory conditions. In practice, due to various reasons and circumstances, such a study is not carried out before the start of treatment, and the doctor is forced to select a drug "at random".

Today, sensitivity testing is performed only in cases where the doctor has serious doubts about whether the prescribed drug will be effective, in cases of prolonged lack of effect from the drug, and also when using the same drug repeatedly in a limited period of time. Sensitivity is often determined in the treatment of sexually transmitted diseases. Many specialists turn to analysis in case of side effects, allergic reactions, and when it is necessary to replace one drug with another.

The analysis is also often used to select drugs for antibacterial therapy in the recovery period after operations, laparoscopic interventions, and organ removal. In departments of surgery and purulent surgery, such a study is simply necessary, since resistance develops here quite quickly. In addition, super-resistant "hospital-acquired" ones develop. Many private clinics approach the prescription of drugs with full responsibility - only after checking sensitivity. In many cases, the budget of state institutions simply does not allow conducting such studies for each patient who requires antibacterial therapy.

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Preparation

Preparation for the study does not require any special measures. It is the same as for any tests. A few days before the study, you should refrain from drinking alcohol. In the morning, on the day of collecting the material, in most cases, you cannot eat or drink. But everything depends on the type of analysis. The material for the study may be different, depending on the disease.

In case of throat and respiratory tract diseases, a throat and nose swab is taken. In venereology, gynecology, urology, genital swabs and blood are taken for analysis. In kidney diseases, urine is often required. In case of gastrointestinal diseases and some infectious diseases, feces and vomit are examined. Sometimes breast milk, nasal discharge, eye secretion, saliva, and sputum can be examined. In case of severe pathologies and suspicion of an infectious process, even cerebrospinal fluid is examined. The spectrum is quite wide.

The features of collecting the material are determined by its biological affiliation. Thus, urine and feces are collected in the morning in a clean container or in a special container for biological material. Breast milk is collected before feeding. The middle portion is taken for examination. The smear is collected using a special swab, which is passed along the mucous membranes, then lowered into a test tube with a prepared medium. Blood is collected in a test tube, from a finger or vein. When collecting smears from the urethra or vagina, it is recommended to refrain from sexual intercourse for several days.

When collecting biological material for research, it is necessary first of all to ensure the correct collection and sterility. But in most cases this is the concern of medical personnel, the patient should not worry about this. Most often, gynecologists and urologists turn to such studies, in second place - otolaryngologists in the treatment of diseases of the nasopharynx and pharynx, upper respiratory tract.

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Technique antibiotic sensitivity testing

The collected biological material is delivered to the laboratory in sterile conditions, where further research is carried out. First of all, its primary seeding is carried out on universal nutrient media. Part of the material is also taken for microscopic examination. A smear for microscopy is prepared, a study is carried out, with the help of which it is possible to determine an approximate picture, to assume which microorganisms are present in the sample. This makes it possible to select the most optimal environments for further research and identification of microorganisms. Also, signs indicating inflammation, an oncological process can be seen on microscopy.

Over the course of several days, colonies of microorganisms grow in a Petri dish. Then, several colonies are taken and transferred to selective nutrient media, which make it possible to determine an approximate group of microorganisms. They are incubated for several days in a thermostat, and then identification (determination of the type of microorganism) begins. Identification is carried out using special biochemical and genetic tests, identifiers. Additionally, immunological studies can be carried out.

After the main pathogen has been isolated, its sensitivity to antibiotics is assessed. There are several methods for this. The most commonly used method is serial dilution, or the disk diffusion method. The methods are described in detail in microbiological reference books, guidelines, and laboratory standards.

The essence of the disk diffusion method is that microorganisms that have been identified are seeded on a nutrient medium, and special disks soaked in antibiotics are placed on top. The seeding is incubated in a thermostat for several days, then the results are measured. The degree of bacterial growth inhibition by each antibiotic is assessed. If the bacterium is sensitive to the antibiotic, a "lysis zone" is formed around the disk, in which the bacteria do not reproduce. Their growth is slow or absent altogether. The diameter of the growth inhibition zone is used to determine the degree of microorganism sensitivity to the antibiotic and formulate further recommendations.

The serial dilution method is the most accurate. For this, microorganisms are seeded on liquid nutrient media, an antibiotic diluted according to the decimal dilution system is added. After this, the test tubes are placed in a thermostat for incubation for several days. Sensitivity to antibiotics is determined by the degree of bacterial growth in a nutrient broth with the addition of antibiotics. The minimum concentration at which microorganisms still grow is recorded. This is the minimum dosage of the drug (recalculation from microbiological units to the active substance is necessary).

These are standard microbiological methods that form the basis of any research. They imply manual execution of all manipulations. Today, many laboratories are equipped with special equipment that performs all these procedures automatically. A specialist working with such equipment only needs the ability to work with the equipment, compliance with safety and sterility rules.

It is necessary to take into account that the sensitivity indices in laboratory conditions and in living organisms differ sharply. Therefore, a person is prescribed a higher dosage than determined during the study. This is due to the fact that the body does not have such optimal conditions for the growth of bacteria. In the laboratory, "ideal conditions" are created. Part of the drug can be neutralized by the action of saliva, gastric juice. Part is neutralized in the blood by antibodies and antitoxins produced by the immune system.

Urine antibiotic sensitivity test

First, biological material is collected. To do this, you need to collect the middle portion of morning urine and deliver it to the laboratory. It is important to maintain sterility and not take antibiotics for several days before the analysis, otherwise you can get a false negative result. After this, a standard sowing is performed, the essence of which is to isolate a pure culture of the pathogen and select an antibiotic that will have an optimal bactericidal effect on it. The required concentration of the antibiotic is determined.

Urine analysis is most often prescribed when there is a suspicion of an infectious and inflammatory process in the genitourinary system, with immunodeficiencies and metabolic disorders. Normally, urine is a sterile liquid. The duration of such a study is 1-10 days and is determined by the growth rate of the microorganism.

Culture and antibiotic sensitivity testing

The study involves isolating the microorganism that is the pathogen into a pure culture. Sometimes there may be several such microorganisms (mixed infection). Some microorganisms are capable of forming biofilms, which are a kind of "microbial communities". The survival rate of biofilms is much higher than that of single microorganisms or associations. In addition, not all antibiotics are capable of influencing the biofilm and penetrating it.

To determine the pathogen, to isolate it into pure culture, sowing is performed. During the study, several sowings are performed in various nutrient media. Then a pure culture is isolated, its biological affiliation is determined, and sensitivity to antibacterial drugs is determined. The optimal concentration is selected.

Any biological material can be used for the study, depending on the disease and localization of the infectious process. The duration is determined by the growth rate of microorganisms.

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Stool sensitivity test

Stool is examined in various gastric and intestinal diseases, in case of suspected infectious process, bacterial intoxication, food poisoning. The purpose of the study is to isolate the pathogen and select optimal antibacterial drugs for it, which will have high activity. The importance of this type of study is that it is possible to select a drug that will affect only the pathogen and will not affect representatives of the normal microflora.

The first and very important stage is collecting feces. It should be collected in a special sterile container in the morning. It should be stored for no more than 1-2 hours. Women with menstrual flow should postpone the analysis until the end, since the accuracy of the results will change. The material is delivered to the laboratory for testing. The analysis is carried out using standard microbiological techniques of sowing and isolating a pure culture. An antibiogram is also carried out. Based on the conclusion, recommendations are developed, and a further study scheme is determined.

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Dysbacteriosis analysis with sensitivity

The material for the study is feces taken immediately after the act of defecation. Normal microflora of the gastrointestinal tract consists of representatives of normal flora and several representatives of pathogenic flora. Their species composition, quantity and ratio are strictly defined and are kept within the permissible norm. If this ratio is disturbed, dysbacteriosis develops. It can manifest itself in different ways. Infectious diseases can develop if the amount of pathogenic microflora increases sharply. If the amount of any microorganism decreases sharply, the free space is occupied by other representatives that are not typical of the gastrointestinal tract, or pathogenic. Often the free space is occupied by a fungus, then various fungal infections and candidiasis develop.

In order to determine the quantitative and qualitative composition of the intestinal microflora, a stool analysis for dysbacteriosis is performed. Conventionally, all representatives living in the intestine are divided into three groups: pathogenic, opportunistic and non-pathogenic. Accordingly, the analysis consists of three parts. Each group of microorganisms has its own needs for a food source, energy. Each group requires separate nutrient media and selective additives.

First, microscopy and primary seeding are performed. Then, after seeding, the largest colonies are selected, similar in morphological features to representatives of each group. They are transferred to selective media. After the microorganisms have grown, they are identified and immediately tested for sensitivity to antibiotics. Standard microbiological methods are used.

The study of a group of pathogenic microorganisms, in addition to standard studies, involves the determination of typhoid, paratyphoid and dysentery bacteria. It is also determined whether a person is a carrier of these microorganisms. A comprehensive study for dysbacteriosis also includes a study of representatives of the bifidobacteria and lactobacilli group. The study takes about a week and depends on the growth rate of the microorganisms.

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Bacteriophage susceptibility testing

In case of intestinal infection, bacteriophages are often used for treatment instead of antibiotics. Bacteriophages are bacterial viruses that are susceptible only to them. They find a bacterium with which they are complementary, penetrate it and gradually destroy the bacterial cell. As a result, the infectious process stops. But not all bacteria are sensitive to bacteriophages. In order to check whether a given bacteriophage will show activity towards representatives of the microflora, an analysis must be carried out.

The material for the study is feces. The analysis must be delivered to the laboratory within an hour, otherwise it will be impossible to conduct. It is necessary to conduct the analysis in several repetitions. The initial method is similar to that for determining sensitivity to antibiotics. First, a preliminary microscopy of the sample is carried out, then primary seeding on universal nutrient media. Then, a pure culture is isolated on selective nutrient media.

The main work is done with pure culture. They are treated with different types of bacteriophages. If the colony dissolves (lyses), this indicates high activity of the bacteriophage. If lysis is partial, the bacteriophage functions moderately. In the absence of lysis, we can talk about resistance to the bacteriophage.

The advantage of phage therapy is that bacteriophages do not affect the human body and do not cause side effects. They attach to certain types of bacteria and lyse them. The disadvantage is that they are very specific and have a selective effect, and cannot always attach to bacteria.

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Sputum analysis for antibiotic sensitivity

The analysis is a study of the discharge from the lower respiratory tract. The goal is to determine the type of microorganisms that act as the causative agent of the disease. An antibiogram is also performed. In this case, the sensitivity of the pathogen to antibiotics is determined, and the optimal concentration is selected. It is used for diseases of the respiratory tract.

The examination of sputum and other contents of the lungs and bronchi is necessary for choosing a treatment regimen and for differentiating various diagnoses. It is used to confirm or refute the presence of tuberculosis.

First, it is necessary to obtain biological material. It can be obtained by coughing, by expectoration, or by taking it from the trachea during bronchoscopy. There are special aerosols that promote expectoration. Before taking sputum, the mouth should be rinsed with water, which will reduce the degree of bacterial contamination of the oral cavity. First, it is recommended to take 3 deep breaths and produce a productive cough. Sputum can also be taken by aspiration from the trachea. In this case, a special catheter is inserted into the trachea. During bronchoscopy, a bronchoscope is inserted into the bronchial cavity. In this case, the mucous membrane is lubricated with an anesthetic.

The material is then sent to the laboratory for testing. Standard seeding and microscopy are performed. A pure culture is then isolated and further manipulations are performed with it. An antibiogram is performed, which makes it possible to identify the spectrum of bacterial sensitivity and select the optimal dosage.

If tuberculosis is suspected, morning sputum is examined for three days. When testing for tuberculosis, the result will be ready in 3-4 weeks. Since mycobacterium tuberculosis, which is the causative agent of the disease, grows very slowly.

Normally, representatives of normal microflora of the respiratory tract should be detected. It is also necessary to take into account that with reduced immunity, the indicators of normal microflora may differ.

Sperm analysis for antibiotic sensitivity

It is a bacteriological study of the ejaculate of sperm with subsequent selection of sensitive antibiotics and their concentrations. Most often it is carried out in the treatment of infertility and other diseases of the male reproductive system. In the event that the disease is accompanied by an infectious process. The main cause of male infertility in most cases is an infection. Usually, a spermogram is initially carried out. Based on the results, the fertilizing ability of the sperm is established. If a large number of leukocytes are found in this analysis, we can talk about an inflammatory process. In this case, a microbiological analysis is usually immediately prescribed, since inflammation is almost always accompanied by an infection. Based on the results obtained, appropriate therapy is selected. The study is usually prescribed by an andrologist.

Prostatitis and venereal diseases are also reasons for conducting the analysis. It is also prescribed if a venereal disease is detected in the partner.

The basis of a correct analysis is, first of all, the correct collection of biological material. The material is collected in special vessels with a wide neck. The storage temperature should correspond to the temperature of the human body. In this case, the material can be stored for no more than an hour. In frozen form, it can be stored for no more than a day. It is inappropriate to take a culture during the intake of antibiotics, this changes the clinical picture. Usually, the culture is taken before the course of antibiotic therapy is started. Or stop taking medications 2-3 days before the analysis.

Then it is sown on a nutrient medium. Incubated in a thermostat for 1-2 days. After that, a pure culture is isolated, then identification is carried out, sensitivity is determined, as well as the type and growth rate of each colony. Sensitivity to antibiotics is determined if pathogenic microorganisms are detected. On average, the analysis takes 5-7 days.

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Gluten sensitivity test

There are many tests that can be used to determine immunological sensitivity to various substances or pathogens. Previously, the main method was to conduct tests based on the agglutination reaction of antibodies and antigens. Today, these tests are used less and less, since their sensitivity is much lower than many modern methods, such as gluten tests. Most often, in practice, they resort to a saliva test for gluten and stool analysis.

The gluten sensitivity test is used to diagnose various intestinal disorders. It is based on the immune system's reaction. If gluten is added to the stool, the reaction occurs or is absent. This is considered a false positive or false negative result. A positive result indicates a predisposition to colitis, a high probability of its development. It also confirms celiac disease.

It is also possible to conduct a gluten test using saliva as a biological material. It is possible to measure the amount of antibodies to gliadin. A positive result indicates sensitivity to gluten. This may indicate a high probability of diabetes. If both tests are positive, diabetes or celiac disease can be confirmed.

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Chlamydia sensitivity test to antibiotics

The analysis is carried out in the treatment of infectious and inflammatory diseases of the urogenital tract, if chlamydia is suspected. The material for the study is a scraping from the vaginal mucosa - in women, a smear from the urethra - in men. The collection is carried out in the procedure room using disposable equipment. It is important to maintain sterility. Before collecting the material, you should refrain from intimacy for 1-2 days before the start of the study. If a woman is menstruating, the material is collected 3 days after its complete end.

The material is delivered to the laboratory. A full analysis includes preliminary microscopy of the smear. This makes it possible to visually determine the microflora by morphological features and correctly select nutrient media. The content of mucus, pus, and epithelial particles may directly or indirectly indicate the development of an inflammatory process or malignant degeneration of cells.

Then, primary seeding is performed. The culture is incubated for several days in a thermostat, and identification is performed based on cultural characteristics. Then, the culture is transferred to selective nutrient media intended for cultivating chlamydia. The resulting colonies are identified using biochemical tests. Then, sensitivity to antibiotics is determined using standard methods. The most sensitive antibiotic and its concentration are selected. Special media developed specifically for this type of microorganism, which contain all the necessary substances and growth factors, are needed to cultivate chlamydia.

It is also possible to conduct a study using a biological method. To do this, rats are infected with the pathogen. In some laboratories, a specially grown tissue culture is used instead of rats. This is due to the fact that chlamydia are intracellular parasites, and special conditions are needed for their cultivation. Then, microorganisms are determined using the PCR method. To determine sensitivity, they are transplanted onto a selective nutrient medium for chlamydia, and after a few days, the results are recorded. Resistance or sensitivity is judged by the suppression of the infectious process in the cells.

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How long does it take to do an antibiotic sensitivity test?

On average, the analysis is done within 5-7 days. Some analyses take longer. For example, when diagnosing tuberculosis, you have to wait from 3 weeks to a month for the results. Everything depends on the growth rate of the microorganisms. Often, laboratory staff have to deal with cases when patients ask for the analysis to be done faster. And they even offer "additional payment" for urgency. However, here you need to understand that in this case, nothing depends on the actions of the laboratory assistant. It only depends on how quickly the microorganism grows. Each type has its own, strictly defined growth rate.

Normal performance

There are no absolute universal norm values for all analyses. Firstly, these values may differ for each biotope. Secondly, they are individual for each microorganism. That is, the norm values for the same microorganism, say, for the throat and intestines are different. So, if staphylococcus predominates in the throat as a representative of normal microflora, then E. coli, bifido- and lactobacteria predominate in the intestines. The values for the same microorganism in different biotopes may also differ significantly. For example, Candida may normally be present in a certain amount in the urogenital microflora. They are not normally present in the oral cavity. The presence of Candida in the oral cavity may indicate their artificial introduction from their natural habitat.

Urine, blood, cerebrospinal fluid are biological environments that should normally be sterile, i.e. should not contain any microflora. The presence of microflora in these fluids indicates a strong inflammatory, infectious process, and also indicates the risk of developing bacteremia and sepsis.

In general, there is an approximate classification. The unit of measurement in microbiology is CFU/ml, that is, the number of colony-forming units in 1 milliliter of biological fluid. The degree of contamination is determined by the number of CFU and varies in a wide range from 10 ¹ to 10 ⁹. Accordingly, 10 ¹ is the minimum number of microorganisms, 10 ⁹ is a severe degree of infection. At the same time, the range of up to 10 ³ is considered normal, all indicators above this number indicate pathological reproduction of bacteria.

As for sensitivity to antibiotics, all microorganisms are divided into resistant, moderately sensitive, sensitive. This result is often expressed as a qualitative characteristic indicating the MID - the minimum inhibitory dose of the antibiotic, which still inhibits the growth of the microorganism. For each person, as well as for each microorganism, these indicators are strictly individual.

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The device for analysis

When conducting bacteriological studies, especially with determination of sensitivity to antibiotics, one device will not be enough. A complete, comprehensive equipment of the bacteriological laboratory is necessary. It is necessary to carefully plan and select equipment that will correspond to each stage of the research. At the stage of collecting biological material, sterile instruments, boxes, boxes, containers, storage chambers and transport equipment for delivering the material to the laboratory are necessary.

In the laboratory, first of all, you will need a high-quality microscope for smear microscopy. Today, there are a large number of microscopes that have a variety of properties - from traditional light to phase-contrast and atomic force microscopes. Modern equipment allows you to scan an image in three-dimensional space and examine it at high magnification with high accuracy.

At the stage of seeding and incubation of microorganisms, autoclaves, dry-heat cabinets, desiccators, steam baths, and a centrifuge may be required. A thermostat is required, in which the main incubation of biological material occurs.

At the stage of microorganism identification and conducting an antibiogram, micromanipulators, mass spectrometers, spectrophotometers, colorimeters may be required for various calculations and assessment of the biochemical properties of cultures.

In addition, modern laboratories can be equipped with high-tech equipment that performs all the above-mentioned main stages of research, right up to calculating the results in automatic mode. Such devices include, for example, a complex device of a bacteriological laboratory based on a time-of-flight mass spectrometer. This line of devices makes it possible to divide the entire territory of the laboratory into three zones. The first zone is dirty, where tests are received and registered. The second zone is the working zone, where the main microbiological research is actually carried out. And the third zone is sterilization and autoclave, where the preparation and disposal of working material is carried out.

The models allow incubation at a wide range of temperatures and conditions. Contains a built-in analyzer of blood and other biological samples, which produces results with high accuracy and reliability. The kit includes electronic scales, bidistillers, centrifuges, autoclaves and sterilization cabinets, automatic medium cooker, water bath with built-in stirrer, pH meters, thermometers and microscopes.

A microbiological analyzer is also used, into which the samples to be tested, nutrient media, and sets of tests to determine sensitivity are placed. The device performs the necessary studies and issues a ready-made conclusion.

Raising and lowering of values

Only a doctor can decipher the analysis. But often, patients, having received the results, panic, noticing a large number of incomprehensible symbols and numbers. In order not to get lost, it is advisable to have at least a general idea of how to decipher the analysis of sensitivity to antibiotics. Usually, the first item in the results indicates the name of the microorganism that is the causative agent of the disease. The name is given in Latin. It can also indicate a representative of the normal microflora that prevails in the body, so there is no need to panic. The second item indicates the degree of seeding, that is, the number of microorganisms. Usually, this number ranges from 10 ¹ to 10 ⁹. The third item indicates the form of pathogenicity, and the fourth - the names of antibacterial drugs to which this microorganism is sensitive. The minimum inhibitory concentration, at which the growth of the microorganism is suppressed, is indicated nearby.

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