RU2626183C1 - Method for determination of sensitivity of obligate anaerobic microorganisms in biofilm to antimicrobial means - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method is proposed to determine the sensitivity of oral microorganisms in biofilms to antimicrobial agents. The method includes culturing of a purulent discharge from the focus of purulent inflammation in the maxillofacial region in the plate wells in a liquid nutrient medium with previously introduced antibiotics under conditions of anaerobiosis and fluid movement. The result is assessed by characteristic signs of biofilm formation, considering the presence of biofilm as a sign of resistance to the antibacterial drug at a given concentration.

EFFECT: method allows to determine the sensitivity of a microorganism in the biofilm to antibiotics without isolation of pure cultures.

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Description

The invention relates to medicine, namely to dentistry, laboratory diagnostics, clinical microbiology, and can be used to determine the sensitivity of microorganisms in a biofilm to antibiotics and other antimicrobial agents in various concentrations when choosing the treatment of purulent-inflammatory diseases of the oral cavity and maxillofacial region ( CHLO).

We know the cassette micromethod for determining the sensitivity of anaerobic bacteria to antibiotics, proposed by us in 1991, which suggested the cultivation of antibiotics in a heated agarized 1.5-2% nutrient medium with the addition of 5% blood and hemin, which was poured into the wells of triacetate microcassettes. Microcassettes were placed in a Petri dish, the inoculum of the studied cultures was seeded and placed in an anaerostat at 37 ° C for 5-7 days. The method is convenient for the cultivation of anaerobic bacteria, including periodontopathogenic group and has been successfully used to date (Tsarev V.N., Ushakov R.V., Pozharskaya V.O., Ippolitov E.V. Principles of antibacterial prevention and therapy in dentistry / in the book: “Microbiology, virology and immunology of the oral cavity (Textbook for dental faculties of medical universities).” - M .: GEOTAR-Media. - 2013. - P. 300-331; Tsarev VN Laboratory diagnosis of anaerobic (clostridial ) infection. / in kN .: "Guide to medical microbiology". // P Dr. Ed Labinskaya AS, Kostyukova NN TH .: Bean -.. 2013 - Book 3 - 1. T. - S. 439-454).

A known method for determining the sensitivity of microorganisms to antibiotics in the treatment of purulent-inflammatory disease (patent RU 2262533 from 10.20.2005, "A method for determining the sensitivity of microorganisms to antibiotics in the treatment of purulent-inflammatory disease", St. Petersburg State Medical University named after Acad. I.P. . Pavlova).

The method includes collecting purulent discharge from the foci of purulent inflammation of the maxillofacial region, culturing in sugar broth for at least two hours, introducing standard discs with the studied antibiotics and incubating for at least two hours. The result is evaluated by the degree of turbidity of the contents in the sample, assuming that the lower the turbidity, the higher the sensitivity. The method allows to determine the sensitivity of the microorganism to antibiotics without isolating a pure culture (that is, in a mixture of different species), that is, with less time.

This system allows you to cultivate a mixture of microorganisms, but also does not allow you to get a biofilm, because it does not provide fluid movement, does not have a solid substrate (glass is not suitable), it is not suitable for reproducing the growth of periodontopathogenic bacteria that require the use of an anaerostat with a specific gas composition therefore, it is not possible to detect the sensitivity to antimicrobial agents of precisely periodontopathogenic microorganisms that do not grow without the formation of a biofilm.

The closest technical solution is the AB-AN test system for determining the sensitivity of obligate anaerobic microorganisms to antibiotics (Kosinets AN, et al. Vitebsk State Medical University, 2011).

A single-use test system "AB-AN" has been developed, which serves to determine the sensitivity of obligate anaerobic microorganisms to antibiotics in a semi-liquid medium after 18-24 hours of incubation under anaerobic conditions. Sensitivity accounting is possible visually or instrumentally using the F300TP enzyme-linked immunosorbent analyzer and a computer with Sensitiv software.

A bacterial suspension is added to the culture medium, which is then introduced into the wells of the tablet containing lyophilized antibiotics (penicillin, amoxicillin + clavulanate, ticarcillin + clavulanate, cefoxitin, imipenem, meropenem, clindamycin, chloramphenicol, metronidazole, amoxicloxin). After incubation, visual or instrumental accounting is performed. Resistant strains grow in the well, causing clouding of the medium; if the strain is sensitive to the antibiotic, the medium remains transparent.

The AB tablet contains 96 wells (12 columns and 8 rows, 2 rows - 24 wells to determine the sensitivity of one strain of the microorganism). In total, the tablet allows you to determine the sensitivity of four microorganisms to 12 antibiotics.

Thus, the system developed by AB-AN is characterized by a wide variety of antibiotics, relative cheapness, ease of manufacture and operation, which will subsequently find wide application for determining the sensitivity of strains of anaerobic pathogens of surgical infections in bacteriological laboratories of various profiles.

However, this system also does not allow culturing the biofilm of anaerobic bacteria and determining the sensitivity of a mixture of microorganisms located on the biofilm.

Thus, the disadvantages of the known methods is the inability to obtain a mixed biofilm from the periodontal pocket or inflammatory focus and to assess the sensitivity of its components (representatives of anaerobic microflora, including periodontopathogenic species) to antibiotics and other antimicrobial agents.

The objective of the invention is to assess the sensitivity to antimicrobial agents of a mixture of microorganisms that form a biofilm isolated from a purulent-inflammatory focus (and located in a mixed biofilm).

The technical result of the proposed method is that due to the creation of gas anaerobic and temperature conditions and oscillatory movements of the nutrient liquid with periodontopathogenic or other anaerobic species in the form of a biofilm in the presence of antibiotics dissolved in a liquid nutrient medium, the effect of accurate specific information on sensitivity obligate-anaerobic microorganisms in a biofilm to antimicrobial agents.

The method allows to determine the sensitivity of the microorganism in the composition of the biofilm to antibiotics without isolating a pure culture, that is, when modeling the conditions of the oral cavity and with less time.

In addition, the determination of the sensitivity of microorganisms to antibiotics in a biofilm is more accurate and correct, since periodontopathogenic species often do not grow as a pure culture without symbiotic microbes. In traditional sowing, as a rule, the growth of precisely the representatives of symbionts is revealed - streptococci, corynebacteria and other representatives of the normal flora and the choice of an antibiotic for the sensitivity of the latter for the treatment of periodontitis is a gross diagnostic error.

When using all the analogues and prototype discussed above, a biofilm does not form and a significant part of microorganisms, including representatives of periodontopathogenic species, die, and other microorganisms remain in the nutrient medium with antibiotic, more often saprophytes and representatives of stabilizing microflora, which play a protective role in the oral cavity. Therefore, the result obtained in the prototype of the result of evaluating sensitivity to microorganisms is not justified for the choice of treatment for purulent-inflammatory diseases of the oral cavity caused by periodontopathogenic species, since it leads to a radically incorrect result of determining sensitivity.

In the inventive method, using a substrate of polymeric materials and oscillatory motions of a liquid (nutrient medium) in which the biofilm is located, it is possible to determine the sensitivity of the entire complex of microorganisms of the biofilm.

The claimed method includes the collection of purulent discharge from the foci of purulent inflammation of the maxillofacial region, culturing it in a liquid nutrient medium containing BHI cardiac broth, or LIM, or AS, 0.01% menadione, 0.1% hemin and an antibacterial drug, on a solid substrate in the wells of a tablet made of polymethyl acrylate or polyurethane or triacetate, while the tablet with the crops is placed in an anaerostat installed in an orbital cultivator that creates the movement of the liquid phase and the formation of a biofilm of microorganisms, Testing is carried out in a mode of 60-120 rocking movements per minute at a temperature of 37 ° C for 2 days, the result is evaluated by the characteristic signs of biofilm formation, considering the lack of biofilm as a sign of resistance to an antibacterial drug at a given concentration.

Further cultivation is carried out under conditions of anaerobiosis (80% nitrogen, 10% carbon dioxide, 10% hydrogen) and the movement of fluid created on an automatic cultivator.

Anaerostat is placed in an orbital shaker-thermostat or cultivator, which creates the movement of the liquid phase of the medium and the formation of a biofilm by microorganisms resistant to this antibiotic and at a given concentration (s). Cultivation is carried out in the mode: 60-120 swinging movements per minute, cultivation temperature 37 ° C, duration - 2 days.

The result is evaluated on a stereomicroscope (or scanning electron microscope) by the characteristic signs of biofilm formation, considering the presence of a biofilm a sign of drug resistance at a given concentration.

Thus, a method has been developed for detecting sensitivity to antimicrobial agents of a complex of microorganisms of the oral cavity in a biofilm, cultivated under strictly anaerobic and temperature conditions on a substrate, immersed in wells with a liquid nutrient medium and an antimicrobial agent under the conditions of oscillatory movements of the platform (i.e., a moving test system), which allows for 48-hour incubation under anaerobic conditions to obtain the full growth of a mixture of microorganisms (including pathogens) in the form of a biofilm and to evaluate the severity ki complex microflora growth of the biofilm by action of the antimicrobial agents studied, planned to treat the patient. Sensitivity accounting is possible visually-instrumental using a stereo microscope or scanning electron microscope, which reduces diagnostic time.

The method is illustrated as follows.

Material intake with a standard cotton swab. The transfer of material on a tampon to the culture medium by mechanical distribution on a substrate or on the bottom of a tablet containing lyophilized antibiotics (optimally 24 drugs used in the treatment of periodontitis and anaerobic infections of the maxillofacial region), where a biofilm is formed under these conditions.

The tablet contains 96 wells (12 columns and 8 rows, 2 rows - 24 holes to determine the sensitivity of the biofilm of one patient). As drugs of choice are used: ampicillin, amoxicillin, amoxicillin + clavulanate, cephalexin, ceftriaxone, cefepime, imipenem, meropenem, lincomycin, clindamycin, chloramphenicol, metronidazole, ciprofloxacin, gemoficyclin cyclin, gemoxifromicin, doxyfromcincyclin, gemoxifromin, azithromycin, fusidine, linezolid).

A total of 96-well plate allows you to determine the sensitivity of the biofilm of 2 patients to 24 antibiotics or make variations in the concentration of drugs according to the traditional dilution scheme to determine the minimum inhibitory concentration (MIC).

If it is necessary to determine the IPC of any antibacterial drug, dilutions of it according to the standard scheme from 125 to 0.12 μg / ml are preliminarily prepared and, accordingly, semi-liquid media with several different dilutions located in decreasing concentration are obtained.

Crops on semi-liquid media containing BHI or LIM cardiac broth or AS, 0.01% menadione, 0.1% hemin and dilutions of the antibacterial drug are placed in an anaerostat with a gas composition: 80% nitrogen, 10% hydrogen and 10% carbon dioxide, which ensures the growth of obligate-anaerobic and microaerophilic species of bacteria belonging to the periodontopathogenic group.

Anaerostat is placed in an orbital shaker-cultivator that creates the movement of the liquid phase of a two-phase medium and the formation of a biofilm by microorganisms resistant to this antibiotic and at a given concentration (s). Cultivation is carried out in the mode: 60-120 swinging movements per minute, cultivation temperature 37 ° C, duration - 2 days.

The results are taken into account using a stereomicroscope or an electronic scanning microscope, which makes it possible to distinguish between the morphology of microbes and the architectonics of a biofilm.

Research results

The presence of biofilm growth on the surface of a dense substrate in a semi-liquid nutrient medium is recorded as follows:

1. In the presence of a mixed biofilm, the microorganisms that make up its composition are considered resistant to this antibacterial drug.

2. In the presence of separate colonies, only these species are considered resistant.

3. In the absence of signs of any growth (biofilm, individual colonies) - all microorganisms of the biofilm are considered sensitive to this antibacterial drug. No growth, the whole mixture is sensitive to antimicrobial agents.

If necessary, to identify the microorganisms that make up the biofilm, conduct further cultural research to determine the biochemical properties of the isolated strains or use modern molecular identification methods (PCR, etc.).

The method was tested on volunteers.

Example No. 1

Patient A. 45 years old, diagnosis: chronic periodontitis of moderate severity, exacerbation phase; periodontal abscess. The purulent contents were taken during the opening of the abscess, inoculation was performed to simulate the biofilm and its sensitivity to 24 different antimicrobial antibiotics was evaluated using the proposed method: ampicillin, amoxicillin, amoxicillin + clavulanate, cefalexin, ceftriaxone, cefepime, imipenemicinfencimenincenomene, meromencimenincenomene, meromenicene, meromencimenomene, meromencimenomene, meromenicene, meromencimenomene, meromencimenincenomene, meromencimenincenomene , metronidazole, ciprofloxacin, levofloxacin, moxifloxacin, hemifloxacin, doxycycline, erythromycin, roxithromycin, spiramycin, clarithromycin, azithromycin, fusidine, linezolid.

Two days later, the results of the study showed that the mixed biofilm formed in the wells of the tablet is present in the wells with the following drugs: ampicillin, amoxicillin, cephalexin, lincomycin, clindamycin, erythromycin, roxithromycin, spiramycin, fusidine (biofilm is stable), but is absent in the holes drugs: amoxicillin + clavulanate, cephalexin, ceftriaxone, cefepime, imipenem, meropenem, chloramphenicol, metronidazole, levofloxacin, moxifloxacin, hemifloxacin, doxycycline, clarithromycin, azithromycin, fusidine, d (biofilm sensitive).

It is recommended to use the combination of amoxicillin + clavunate and metronidazole for treatment.

Example No. 2

Patient M., 26 years old, diagnosed with aggressive generalized periodontitis. Unsuccessful antibiotic treatment of the group of linkosamides (lincomycin) did not give positive results, and we evaluated the sensitivity of the biofilm of the periodontal (gingival) pocket of the patient to 24 antibiotics.

Two days later, the results of the study showed that the mixed biofilm formed in the wells of the tablet is present in the wells with the following drugs: ampicillin, amoxicillin, amoxicillin + clavulanate, cephalexin, ceftriaxone, cefepime, lincomycin, clindamycin, metronidazole, ciprofromycincin docycloxincycincincin , spiramycin, clarithromycin, clarithromycin (biofilm is stable), but is absent in the wells with drugs: imipenem, meropenem, chloramphenicol, levofloxacin, moxifloxacin, gemifloxacin, linezolid (biofilm and sensitive).

It is recommended for treatment to use one of the fluoroquinolone drugs of the 3rd generation (levofloxacin) or 4 generations (moxifloxacin, hemifloxacin).

In addition, in patient M., a study of the MPC of a group of fluoroquinolones, gatifloxacin, was conducted. To determine the MPC from a sample of the preparation, dilutions were prepared according to the standard scheme in the range from 125 μg to 0.12 μg / ml, which were added to the wells in a plastic tablet and dried so that the final concentration in the wells when adding the nutrient medium was from 125 μg / ml to 0.12 μg / ml. Then, a three-species biofilm was inoculated, including Streptococcus sanguis, Fusobacterium nucleatum, Porphyromonas gingivalis, filled with a nutrient medium and after 48 hours of cultivation under specified conditions (temperature, anaerobiosis, movement of the liquid phase of the medium), the result was taken into account using a stereo microscope.

It was found that in the range from 125 μg to 0.48 μg / ml, biofilm growth was absent. At a concentration of 0.24 μg / ml, individual biofilm fragments represented by streptococci (Streptococcus sanguis) were observed, and at a concentration of 0.12 μg / ml, a pronounced growth of a three-species biofilm was observed.

Therefore, the MPA of gatifloxacin (the minimum concentration at which microbial growth is completely absent) in relation to periodontopathogenic biofilm was 0.48 μg / ml.

Thus, the developed proposed method for determining the sensitivity of obligate-anaerobic microorganisms to antimicrobial agents in a mixed biofilm makes it possible to study any variety of antibiotics or other antimicrobial agents, the claimed method illustrates the high accuracy and specificity of obtaining medical information about the studied antimicrobial agent, its suitability for treating a patient in short terms (2 days instead of a week), ease of manufacture and operation, which will allow further Iti widespread use of this method for determining the sensitivity of strains of anaerobic and anaerobic periodontal pathogens infections in bacteriology laboratories different profile. Given the important role of periodontopathic anaerobic microorganisms in the pathogenesis (development) of not only oral diseases, but also cardiovascular and gastrointestinal pathologies, the method can be used in laboratory diagnosis of the choice of drugs by various departments of the clinic.

Claims (1)

A method for determining the sensitivity of periodontopathogenic obligate-anaerobic microorganisms in a biofilm to antimicrobial agents, including collecting purulent discharge from the site of purulent inflammation of the maxillofacial region, culturing it in a liquid nutrient medium containing BHI cardiac broth, or LIM, or AS, 0.01 % menadione, 0.1% hemin and an antibacterial drug, on a solid substrate in the wells of a tablet made of polymethyl acrylate, or polyurethane, or triacetate, while the tablet with crops is placed in an anaerostat, installed th into the orbital cultivator, which creates the movement of the liquid phase and the formation of a biofilm of microorganisms, and the cultivation is carried out in the mode of 60-120 rocking movements per minute at a temperature of 37 ° C for 2 days, the result is evaluated by the characteristic signs of biofilm formation, considering the presence of a biofilm a sign of resistance to antibacterial drug at a given concentration.