Analysis for sensitivity to antibiotics: preparation, decoding, how much is done

Today, the analysis of sensitivity to antibiotics is becoming increasingly popular. The microflora of man 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 allow maintaining a normal ratio and the number of microbial populations. Since the beginning of the era of antibiotics, many diseases that were previously considered fatal have been cured. But microorganisms also tend to survive, gradually adapting to the action of antibacterial drugs. Over time, many of them became resistant to many medicines, consolidated it in the genotype and began to pass from generation to generation. Thus, new microorganisms are already not initially sensitive to certain drugs, and their purpose may be ineffective. Pharmacists are developing more and more new products, adding new active ingredients to them, changing the basic formula. But gradually, they also develop resistance.

The reason for the increased resistance of microflora to many drugs, and even their analogues is often hidden in the wrong and uncontrolled intake 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 chosen, which is very important both for treatment and for preventing mechanisms for the development of further resistance. Many mistakenly prescribe antibacterial therapy even in viral diseases, which is ineffective, since the antibiotic does not act against the 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 "in the blind", there are often cases when they show no activity towards those microorganisms that caused the disease and whose number should be reduced. Instead, they affect other representatives of the microflora, resulting in a dysbiosis, which is also a rather dangerous pathology and can lead to serious consequences. Especially dangerous are cases when the antibiotic destroys the normal microflora, which is designed to protect the body and maintain its normal functioning. Also, there are cases when too much or too little dosage is prescribed.

Patients also are irresponsible to treatment. Often, treatment is discontinued after the symptoms stop worrying. At the same time, many people prefer to complete the full course until the end. This is one of the factors that contribute to the development of resistance in bacteria. The full course is designed to completely 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 remedy, and pass it on to the next generations. The danger is that resistance is developed not only in relation to this particular drug, but also to the entire group of drugs.

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

[1], [2], [3], [4], [5], [6], [7], [8]

Indications for the procedure of the antibiotic susceptibility testing

Normally, such an analysis should be carried out in all cases when antibacterial therapy is required. Proceeding from 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 made, in the laboratory conditions the optimal concentration of the active substance has been determined. In practice, due to various reasons and circumstances, such a study before the start of treatment is not performed, and the doctor is forced to select a drug "at random".

Today, sensitivity testing is performed only in those cases when the doctor has great doubts as to whether the intended remedy will be effective, in cases of prolonged absence of the effect of the drug, as well as in the repeated use of the same agent in a limited period of time. Often, sensitivity is 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.

They are also often referred to for the selection of medicines for antibiotic therapy during the recovery period after surgery, laparoscopic interventions, and removal of organs. In surgical surgeries, purulent surgery, such a study is simply necessary, since resistance develops quite rapidly here. In addition, super-stable "hospital-acquired" ones are developing. Many paid clinics approach the prescription of medicines with all responsibility - only after testing the sensitivity. In many cases, the budget of public institutions simply does not allow such studies to be carried out for every patient who needs antibiotic therapy.

[9], [10], [11]

Preparation

Preparation for the study does not require any special measures. It is the same as in any analysis. A few days before the study you need to refrain from drinking alcohol. In the morning, on the day of sampling, in most cases, you can not eat and drink. But it all depends on the type of analysis. The material for the study may be different, depending on the disease.

With diseases of the throat, respiratory tract, take a swab from the throat, nose. In venereology, gynecology, urology, take for analysis swabs from the genitals, blood. With kidney disease, urine is often required. With diseases of the digestive tract, some infectious diseases, examine the excrement, vomit. Sometimes breast milk, nasal discharge, eye secretions, saliva, sputum may be investigated. In severe pathologies and suspicion of the infectious process, even spinal fluid is examined. The spectrum is wide enough.

The nature of the material intake is determined by its biological affiliation. So, urine, feces, is collected in the morning in a clean container or in a special container for biological material. The collection of breast milk is performed prior to feeding. The study takes an average portion. The smear is taken with a special tampon, which is carried on the mucous membrane, then lowered into a test tube with a prepared medium. Blood is collected in a test tube, from a finger or vein. When taking swabs from the urethra or vagina, it is recommended to abstain from sexual intercourse for several days.

When collecting biological material for research, it is first necessary to ensure the correctness of the fence and sterility. But this in most cases is the care of medical personnel, the patient should not be worried about this. Most often, gynecologists and urologists turn to similar studies, in second place - otolaryngologists in the treatment of diseases of the nasopharynx and pharynx, upper respiratory tract.

[12], [13], [14]

Technique of the antibiotic susceptibility testing

The collected biological material under sterile conditions is delivered to the laboratory, where further investigations are carried out. Primarily, its primary seeding is performed on universal nutrient media. Also part of the material for microscopic examination is taken. A smear for microscopy is being prepared, a study is being conducted, by means of which an approximate picture can be determined, suggesting which microorganisms are present in the sample. This makes it possible to fit the most optimal environments for further research and identification of microorganisms. Also, on the microscopy, there may be signs that indicate inflammation, an oncological process.

For several days in the Petri dish, colonies of microorganisms grow. Then, several colonies are taken, they are crossed into selective nutrient media, which make it possible to determine an approximate group of microorganisms. Incubate for several days in a thermostat, then proceed to identify (determine the type of microorganisms). Identification is carried out with the help of special biochemical and genetic tests, determinants. In addition, immunological follow-up may be performed.

After the main pathogen is isolated, conduct an assessment of its sensitivity to antibiotics. There are several methods for this. The most commonly used method of serial dilution, or disk-diffusion method. Techniques are detailed in microbiological reference books, methodological guidelines and laboratory standards.

The essence of the disco-diffusion method consists in the seeding of microorganisms that have been identified on the nutrient medium, special discs impregnated with antibiotics are superimposed on top. Within a few days, the crop is incubated in a thermostat, then the results are measured. Estimate the degree of retardation of bacterial growth by each antibiotic. If the bacterium is sensitive to the antibiotic, a "lysis zone" is formed around the disc, in which the bacteria do not reproduce. Their growth is slow, or completely absent. The diameter of the growth retardation zone is determined by the degree of sensitivity of the microorganism to the antibiotic and formulates further recommendations.

The serial dilution method is the most accurate. To do this, microorganisms are sown on liquid nutrient media, add an antibiotic diluted in the system of decimal dilutions. After this, the tubes are placed for several days in a thermostat for incubation. Sensitivity to antibiotics is determined by the degree of growth of bacteria in the nutrient broth with the addition of an antibiotic. Record the minimum concentration at which the growth of microorganisms is still occurring. This is the minimum dosage of the drug (recalculation from microbiological units to the active substance is necessary).

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

It should be taken into account that the sensitivity indices in the laboratory and in the conditions of a living organism are sharply different. Therefore, a higher dosage is prescribed to a person than was 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. The part is neutralized in the blood by antibodies and antitoxins, which are produced by the immune system.

Urine analysis for sensitivity to antibiotics

To begin with, biological material is collected. To do this, you need to collect an average portion of the morning urine and deliver it to the laboratory. It is important to observe sterility and do not take antibiotics several days before the analysis, otherwise you can get a false negative result. After this, a standard crop is produced, the essence of which consists in isolating the pure culture of the pathogen and selecting an antibiotic that will have the best bactericidal effect on it. The necessary concentration of antibiotic is determined.

The analysis of urine is most often prescribed for suspected 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.

Analysis for culture and sensitivity to antibiotics

The study implies the isolation of a microorganism, which is the causative agent in a pure culture. Sometimes such microorganisms can be several (mixed infection). Some microorganisms are able to form biofilms, which are peculiar "microbial communities". Survival of biofilms is much higher than single microorganisms, or associations. In addition, not all antibiotics are able to affect the biofilm, and penetrate it.

 To determine the pathogen, its isolation into a pure culture, seeding is carried out. During the research, several crops are cultivated, in various nutrient media. Then a pure culture is allocated, biological significance is determined, and susceptibility to antibacterial drugs is determined. The optimal concentration is selected.

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

[15], [16], [17], [18], [19], [20]

Fecal analysis for sensitivity

The feces are examined for various gastric and intestinal diseases, with suspicion of an infectious process, bacterial intoxication, food poisoning. The aim of the study is to isolate the pathogen and to select the optimal antibacterial drugs that will be highly active. The importance of this type of study is that it is possible to select a drug that will only affect the causative agent of the disease, and will not affect the representatives of normal microflora.

The first and very significant stage is the collection of stool. It must be collected in a special sterile container, in the morning hours. Keep no more than 1-2 hours. Women with menstrual flow should postpone the analysis until the end, because the accuracy of the results will change. The material is delivered to the laboratory for examination. The analysis is carried out using the standard microbiological technique of inoculation and the isolation of pure culture. Additionally, an antibioticogram is performed. According to the conclusion, recommendations are developed, further research scheme is determined.

[21], [22], [23], [24]

Analysis for dysbiosis with sensitivity

The material for the study is the feces taken immediately after the act of defecation. Normal gastrointestinal microflora consists of representatives of normal flora and several representatives of pathogenic flora. Their species composition, quantity and correlation are strictly marked and kept within the permissible norm. If such a ratio is violated, dysbacteriosis develops. It can manifest itself in different ways. Infectious diseases can develop if the amount of pathogenic microflora sharply increases. If the quantity of any microorganism decreases drastically, other representatives occupy a free place, which are not characteristic of the gastrointestinal tract, or pathogenic. Often an empty seat is occupied by a fungus, then various fungal lesions develop, candidiasis.

In order to determine the quantitative and qualitative composition of the intestinal microflora, the feces are analyzed for dysbacteriosis. Conditionally, all representatives of 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 needs in the source of nutrition, energy. For each group, separate nutrient media and selective additives are needed.

First, microscopy and primary seeding are carried out. After the sowing, the largest colonies are selected, morphologically similar to the representatives of each group. Produced by re-settling on selective media. After the microorganisms have grown, they are identified and immediately tested for antibiotic sensitivity. Standard microbiological methods are used.

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

[25], [26], [27], [28], [29]

Analysis for sensitivity to bacteriophages

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

The material of the study is cal. The analysis should be delivered to the laboratory within an hour, otherwise it will not be possible. It is necessary to carry out the analysis in several replicates. The original technique is similar to that in determining sensitivity to antibiotics. First, a preliminary microscopy of the sample is carried out, then primary seeding on universal nutrient media. Then, a selective culture is produced on selective nutrient media.

The main work is conducted with pure culture. They are treated with various types of bacteriophages. If the colony dissolves (lysed), this indicates a high activity of the bacteriophage. If the lysis occurs partial - the bacteriophage functions moderately. In the absence of lysis, one can speak of resistance to a bacteriophage.

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

[30], [31], [32], [33], [34], [35], [36], [37], [38]

Sputum analysis for sensitivity to antibiotics

The analysis is a study of the detachable lower respiratory tract. The aim is to determine the type of microorganisms that act as a causative agent of the disease. An antibioticogram 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 respiratory diseases.

Examination of sputum and other contents of the lungs and bronchi is necessary for choosing a therapy scheme, for differentiating different diagnoses. It is used to confirm or deny the presence of tuberculosis.

First you need to get biological material. It can be obtained by coughing, by expectoration, or by fetching from the trachea with bronchoscopy. There are special aerosols that contribute to 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, to 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. When bronchoscopy is introduced into the cavity of the bronchus bronchoscope. In this case, the mucous membrane is lubricated with an anesthetic.

Then the material is delivered to the laboratory for study. Sowing is carried out according to the standard scheme, microscopy. Then a clean culture is isolated, and further manipulations are carried out with it. An antibioticogram is placed, which makes it possible to identify the spectrum of bacterial sensitivity and to select the optimal dosage. 

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

Normally, representatives of the normal microflora of the respiratory tract should be found. Also it is necessary to take into account that with reduced immunity the parameters of normal microflora may differ.

Analysis of sperm sensitivity to antibiotics

It is a bacteriological study of sperm ejaculate with further 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 is in most cases the infection. Usually a spermogram is performed initially. By results, the fertilizing capacity of the sperm is determined. If this analysis shows a large number of white blood cells, we can talk about the inflammatory process. At the same time, microbiological analysis is usually prescribed immediately, since inflammation is almost always accompanied by an infection. Based on the results obtained, appropriate therapy is selected. The study is usually prescribed by the andrologist.

Also, the reason for the analysis is prostatitis, venereal diseases. Assign and in the event that a partner has a sexually transmitted disease.

The correct analysis is based, first of all, on the correct selection of biological material. Take the material in special vessels with a wide throat. 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 can be stored no more than a day. During the reception of antibiotics, the sowing is inadvisable, this changes the clinical picture. Usually, the crop is surrendered before the course of antibiotic therapy is started. Or stop taking medication 2-3 days before the test.

Then, it is sown on a nutrient medium. Incubate in the thermostat for 1-2 days. After a clean culture is isolated, then the identification is carried out, the sensitivity is determined, and the type and growth rate of each colony. Sensitivity to antibiotics is determined in the case of detection of pathogenic microorganisms. On average, the analysis is done 5-7 days.

[39], [40], [41], [42], [43],

Analysis for sensitivity to gluten

There are many tests with which you can determine the immunological sensitivity to various substances or pathogens. Previously, the main method was to perform tests based on the agglutination reaction of antibodies and antigens. Today, these tests are used less and less, because their sensitivity is much lower than many modern techniques, for example, gluten tests. Most often in practice resort to a saliva test for gluten and analysis of feces.

The gluten sensitivity test is used to diagnose various disorders of the intestine. It is based on the reaction of the immune system. If gluten is added to the stool, the reaction occurs, or is absent. This is regarded as a false positive or false negative result. Positive indicates a predisposition to colitis, a high probability of its development. Also confirms celiac disease.

Gluten can also be analyzed using saliva as a biological material. You can measure the amount of antibodies to gliadin. A positive result indicates a sensitivity to gluten. This may indicate a high probability of diabetes. If the result is positive in both tests, you can confirm iabet or celiac disease. 

[44], [45]

An analysis of the sensitivity of chlamydia to antibiotics

The analysis is carried out in the treatment of infectious and inflammatory diseases of the urogenital tract, with suspicion of chlamydia. The material for the study is scraping from the vaginal mucosa - in women, a smear from the urethra - in men. The fence is made in the treatment room using disposable equipment. It is important to observe sterility. Before taking the material, you should refrain from intimacy within 1-2 days before the start of the study. If a woman has menstruation, the material is taken 3 days after her complete termination.

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

Then the primary crop is produced. The culture is incubated for several days under a thermostat, and is identified by culture. Then it is produced by re-settling on selective nutrient media intended for the cultivation of chlamydia. The resulting colonies are identified using biochemical tests. After determining the sensitivity to antibiotics by standard methods. Choose the most sensitive antibiotic, its concentration. For the cultivation of chlamydia, special media are needed, designed specifically for this type of microorganism, which contain all the necessary substances and growth factors.

It is also possible to conduct a biological study. To do this, infect the causative agent of rats. 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 for their cultivation special conditions are needed. The microorganisms are then determined by the PCR method. To determine the sensitivity, a transplant is made to a selective culture medium for chlamydia, after a few days the results are recorded. On resistance or sensitivity is judged by the suppression of infection in cells.  

[46], [47], [48], [49], [50], [51], [52], [53], [54]

How much is the antibiotic sensitivity test done?

On average, the analysis is done within 5-7 days. Some tests are done longer. For example, when diagnosing tuberculosis, the results should be expected from 3 weeks to a month. Everything depends on the growth rate of microorganisms. Often, laboratory staff have to deal with cases where, patients are asked to do the analysis faster. And they even offer a "surcharge" for urgency. However, here it is necessary to understand that nothing depends on the activities of the laboratory assistant in this case. And it depends only on how quickly the microorganism grows. Each species has its own, strictly defined growth rate.

Normal performance

There are no indicators of an absolute universal standard for all analyzes. First, for each biotope these indicators may differ. Secondly, they are individual for each microorganism. That is, the indicators of the norm of the same microorganism, say, for the throat and intestine differs. So, if the staphylococcus predominates in the throat as a representative of normal microflora, then the intestine is dominated by E. Coli, bifido- and lactobacilli. Also, indicators for the same microorganism in different biotopes may differ significantly. For example, candida can normally be contained in a certain amount in the urogenital microflora. In the oral cavity, they are not normally contained. The penetration of candida into the oral cavity may indicate their artificial drift from their natural habitat.

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

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

As for sensitivity to antibiotics, all microorganisms are divided into stable, moderately sensitive, sensitive. Often this result is expressed as a qualitative characteristic with indication of MIG - the minimal inhibitory dose of an antibiotic that still inhibits the growth of the microorganism. For each person, as for every microorganism, these indicators are strictly individual.

[55], [56], [57], [58]

The device for analysis

When carrying out bacteriological studies, especially with the definition of sensitivity to antibiotics, one apparatus will not be enough. A full, comprehensive equipment of the bacteriological laboratory is necessary. It is necessary to carefully plan and select the equipment that will correspond to each stage of research. At the stage of sampling of biological material, sterile instruments, boxes, bixes, containers, storage chambers and transportation equipment are needed to deliver material to the laboratory.

First of all, a high-quality microscope for smear microscopy is needed in the laboratory. Today, there is a large number of microscopes, which have a variety of properties - from the traditional light to the phase-contrast and atomic force microscope. Modern equipment allows you to scan an image in three-dimensional space and view it at high magnification with high accuracy.

At the stage of planting and incubation of microorganisms autoclaves, dry-fire cabinets, desiccators, steam baths, and a centrifuge may be required. A thermostat is essential, in which the main incubation of biological material takes place.

At the stage of identification of microorganisms and the conduct of an antibioticogram, micromanipulators, mass spectrometers, spectrophotometers, colorimeters for various counts and estimates of the biochemical properties of cultures may be required.

In addition, modern laboratories can be equipped with high-tech equipment that performs all of the above main stages of the investigation, up to the calculation of results in an automatic mode. Among 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 laboratory into three zones. The first zone is dirty, in which the analysis is taken, registration. The second zone is a working zone, in which, in fact, they make basic microbiological studies. And the third zone - sterilization and autoclave, where the preparation and utilization of working material is carried out.

Models make it possible to incubate under a wide range of temperatures and conditions. The built-in analyzer of blood and other biological samples contains the results with high accuracy and reliability. Electronic scales, bidistillators, centrifuges, autoclaves and sterilization cabinets, automatic media, water bath with built-in stirrer, pH-meters, thermometers and microscopes are included.

Also, a microbiological analyzer is used, in which test samples, nutrient media, test sets for determining sensitivity are laid. The apparatus carries out the necessary studies and issues a ready-made conclusion.

Raising and lowering of values

Decoding of the analysis can only be done by a doctor. But often patients, having received a result in their hands, panic, noticing a large number of incomprehensible symbols and figures. In order not to be lost, it is advisable to have at least a general idea of how to decipher the analysis for sensitivity to antibiotics. Usually in the results, the first item indicates the name of the microorganism, which is the causative agent of the disease. The name is in Latin. Also, a representative of normal microflora that predominates in the body can be indicated here, so do not panic. The second item indicates the degree of seeding, that is, the amount of microorganism. 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 next.

[59], [60], [61], [62], [63], [64]