RU2417376C2 - METHOD FOR ASSESSING CLINICAL EFFICACY OF ANTIBACTERIAL PREPARATIONS FOR SPECIAL DANGER INFECTIOUS AGENTS Francisella tularensis AND Brucella spp - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: assessment of the clinical efficacy of antibacterial preparations is three-staged; the first stage involves working up an antibiotic-sensitivity criteria scale for reference strains; at the second stage the antibiotic-sensitivity of an investigated material is evaluated, while the third stage consists in correlating the latter values with the derived scale for a given type of microbe; the first and second stages are simultaneous and based on the same technique by inoculation of a microbial suspension in the amount 0.3 ml (n·10⁷CFU/ml) by a diffusion test and a dilution test on a nutrient medium with growth supplements. Thereafter, the inoculation is incubated at 37°C for 24-48 hours, and the results are analysed. The assessment of the clinical efficacy is enabled by correlating the border values of minimal inhibitory concentration and growth inhibition zone diametres with the relevant criteria scale. If the values of the investigated material fall within a permissible range of the criteria scale, an antibiotic is considered to be effective.

EFFECT: use of the method enables the reliable and immediate assessment of the clinical efficacy of antibacterial preparations for tularemic and brucellous infections.

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Description

The invention relates to medicine, namely to medical microbiology, and can be used to treat diseases caused by pathogens of especially dangerous infections, in particular when choosing the most effective treatment regimens for tularemia and brucellosis.

Tularemia and brucellosis are classified as highly dangerous infectious diseases of humans and mammals, endemic foci of which are widespread in various countries, including Russia.

The natural resistance of Francisella tularensis to penicillins, cephalosporins, macrolides and polymyxin is known, which significantly limits the choice of etiotropic therapy for infections. In addition, brucellosis is extremely poor in antibiotic therapy.

Therefore, the assessment of the sensitivity of these pathogens to antibacterial drugs is an important step in determining the effectiveness of etiotropic therapy.

Known methods for determining the sensitivity of microorganisms to antibacterial drugs (see Guidelines MUK 4.2.1890-04, Moscow, 2004, p. 47), including determining the sensitivity of microorganisms with complex nutritional needs using the serial dilution method and the disk diffusion method for Hinton-Muller environment.

However, a disadvantage of the known method is that the antibiotic susceptibility medium proposed by the MUK for bacteria (Haemophilus, Pneumococcus, Neisseria) does not ensure the growth of tularemia microbe and brucella within 24-48 hours. In this regard, the above criteria for assessing the sensitivity of resistance of whimsical or unpretentious microorganisms do not meet the criteria for assessing the antibiotic sensitivity of especially dangerous pathogens, such as francicella or brucella.

For the prototype, a method was selected for predicting the clinical efficacy of an antibacterial agent by the dilution method (see US Pat. RU 2192642, class Q01N 33/569, 2000.09.07), including incubation of a suspension of microbial cells with an antibacterial agent in tablet cells, which are taken in two concentrations, corresponding to those that are created in target organs with the introduction of medium and higher therapeutic doses, the results are recorded visually by the appearance of visible growth in the cells of the tablet.

The disadvantage of the prototype is that for a correct assessment, it is necessary to carry out a control seeding from a clouded broth to exclude contamination by extraneous microflora, which extends the method accounting time by another 24 hours.

In addition, when working with pathogens of especially dangerous human infections, namely the causative agents of tularemia and brucellosis in accordance with SP 1.3.1285-03.2003 “Safety of work with microorganisms of pathogenicity groups I and II”, open polystyrene plates are not allowed.

At the same time, in order to choose the concentrations corresponding to the average therapeutic and highest doses, it is necessary to have the appropriate tables, which are not available in laboratory practice, therefore, in the event of an epidemic outbreak, the need to search for such information will require additional time to produce reliable results.

The technical task of the invention consisted in the development of a method that reliably and quickly obtain an assessment of the clinical effectiveness of antibacterial drugs in tularemia and brucellosis infections.

This goal is achieved by the fact that in the known method for evaluating the clinical effectiveness of antibacterial drugs for pathogens of especially dangerous infections Francisella tularensis and Brucella spp., Which includes the cultivation of microbial cells in a nutrient medium using methods: disk diffusion and serial dilutions, and the assessment is carried out in three stages, according to the first stage, a scale of antibiotic sensitivity criteria for reference strains is developed, the second determines the sensitivity of the test material to the same antibiotics, and the third for It becomes active as compared with the obtained latest performance scale for this type of microbe, wherein the first and second steps are carried out simultaneously on the same technology by inoculating the microbial suspension in a volume of 0.3 ml of (n · July ¹⁰ cfu / ml) for disc-diffusion method and the method serial dilutions in a nutrient medium with growth additives, after inoculation, they are incubated at 37 ° C for 24-48 hours and the results are recorded, and clinical efficacy is assessed by comparing the boundary values of the minimum inhibitory concentration and diameter s suppression of pathogen growth areas with the appropriate criteria for the scale, if the performance of the material included in the allowable range of the criteria of the scale, the antibiotic is considered effective.

In addition, when creating the criteria scale for Francisella tularensis, subsp strains are used. nearctica., mediasiatica, holarctica.

At the same time, developing a scale of criteria for Brucella spp., Three types of strains are used: B. abortus, B. suis, B. melitensis.

In addition, as growth additives to Muller-Hinton agar, components containing in g / l of medium are used:

magnesium sulfate - 0.05;

iron sulfate - 0.1;

L-cysteine - 0.6;

lemon sodium - 1.2;

potassium chloride - 2;

potassium phosphate - 4;

glucose - 15.

The proposed method is carried out in the following steps:

Stage I - develop a scale of criteria for assessing antibiotic sensitivity for the reference strains of Francisella tularensis and Brucella spp .;

Stage II - assess the sensitivity of the investigated material (culture) to antibacterial drugs;

Stage III - carry out a comparative analysis of the results of the studied material (stage II) with a scale of criteria (stage I) to assess the clinical effectiveness of antibacterial drugs.

For the development of stage I, reference strains of tularemia microbe of three main subspecies (F. tularensis subsp. Nearctica, F. tularensis subsp. Mediasiatica, F. tularensis subsp. Holarctica) and strains of the pathogen of brucellosis of three species (B. abortus, B. suis, B . melitensis) (see tables 1, 3).

When carrying out the 1st and 2nd stages, Müller-Hinton agar is used as the optimal nutrient medium with the addition of the following growth components containing in g / l of medium: magnesium sulfate - 0.05; iron sulfate - 0.1; L-cysteine - 0.6; sodium citrate - 1.2; potassium chloride - 2; potassium phosphate - 4; glucose - 15, pH 7.3.

Antibiotics are used in the evaluation method: the first row (doxycycline, ciprofloxan, ofloxacin, norfloxacin, lomefloxacin, levofloxacin, streptomycin, gentamicin, rifampicin) and the second row (amikacin, kanamycin, nalidixic acid, tetracycline).

The sensitivity of microorganisms to antibiotics is determined using the disk diffusion method (DDM) and the method of serial dilutions (MCP). For the latter method, in a dense nutrient medium, a series of doubly increasing concentrations of antibacterial drugs is prepared, for example: doxycycline 1-2-4-8-16, ciprofloxacin 0.06-0.12-0.25-0.5-1.

To determine antibioticograms of bacteria using the disk diffusion method, proven commercial discs with specific concentrations of antibacterial drugs are used.

Steps 1 and II are carried out using the same technology.

When determining the sensitivity using DDM, a suspension of microbial cells in a 0.85% sodium chloride solution prepared from a 24 h agar culture of the studied bacteria is used.

Suspension

The suspension is standardized according to the industry standard turbidity GISK them. L.A. Tarasovich for 5 units. (n · 10 ⁸ CFU / ml). The suspension is applied to the surface of the agar with growth additives in a volume of 0.3 ml (n · 10 ⁷ CFU / ml), evenly distributed with a spatula and incubated until completely absorbed into the agar. After this, discs are applied (no more than 6 per cup with a diameter of 100 mm). Crops are incubated at 37 ° C for 24-48 hours. The results are taken into account only in the event of drainage growth on control plates (without discs). The diameter of the zones of growth inhibition of bacteria around the discs (including the diameter of the disc itself) is measured with an accuracy of 1 mm with a ruler-piece or vernier caliper (see table 1, 3).

For MCP, a suspension of the same concentration (n · 10 ⁷ CFU / ml) is used, which is applied in small drops using a replicator or by pipetting onto plates with growth medium supplemented with various concentrations of antibacterial drugs, starting with a plate with a minimum content antibiotic. Crops are incubated at 37 ° C for 24-48 hours. The results are taken into account in the presence of culture growth on control plates (without antibiotic).

The sensitivity / resistance of cultures is evaluated by the minimum concentration of the drug, inhibiting the growth of microbes (MIC, μg / l = mg / l) (see table 2, 4).

In stage III, a comparative analysis of the indicators (IPC and zone diameter) of the test material is carried out with a scale of criteria developed for Francisella tularensis (table 2) and for Brucella spp. (table 4).

The sensitivity of the strains of tularemia and brucellosis microbe is assessed in accordance with the boundary values of the IPC and the diameters of the growth inhibition zones for sensitive and resistant crops (see table 2, 4), if the parameters of the studied material are included in the acceptable range of the criteria scale, then the antibiotic is considered effective in view that bacteria are sensitive to the proposed antibiotic.

An increase in the IPC values of drugs or a decrease in the diameters of zones of inhibition of growth of the studied material in comparison with these indicators for control strains (criteria scale) indicate a tendency to increase the resistance of microorganisms and will require the use of maximum therapeutic doses, prolongation of their use or the use of another antibacterial drug.

The use of the method for assessing the clinical effectiveness of antibacterial drugs in tularemia and brucellosis infections is confirmed by examples.

Example 1

From the water of the Don River, a culture was identified, later identified as Francisella tularensis (P-13862). A study was made of the resistance of the isolated culture of tularemia microbe to first-line drugs - aminoglycosides (gentamicin), fluoroquinolones (ciprofloxacin), doxycycline and rifampicin using the disk diffusion method and the serial dilution method in Hinton-Muller agar with growth additives. Analysis was carried out after 24 and 48 hours. When using the disk diffusion method, the studied pathogen culture had the following indicators (bacterial growth inhibition zone): for gentamicin — 27 mm, for ciprofloxacin — 36 mm, for rifampicin — 32 mm, doxycycline — 30 mm. When using the serial dilution method in agar, the MPC of antibiotics was (g / l): for gentamicin - 0.5, ciprofloxacin - 0.06, rifampicin - 0.125, doxycycline - 1. In accordance with the criteria developed by the RFI for assessing the resistance / sensitivity of the tularemia pathogen to antibacterial drugs (see table 2) the studied culture is sensitive to these antibiotics. The results obtained (see table 5) made it possible to recommend these antibiotics for the treatment of people with tularemia.

Example 2

When an employee worked in a laboratory with three experimental strains of tularemia microbe with markers of resistance to nalidixic acid, gentamicin or rifampicin and doxycycline, a situation arose that suggests the possibility of human infection. When studying the sensitivity of strains to first-line antibiotics using DDM, the following indicators were obtained (zone of growth inhibition of bacteria): for gentamicin - 16 mm, ciprofloxacin - 10 mm, rifampicin - 32 mm, doxycycline - 30 mm. When using the serial dilution method in agar, the MPC of antibiotics was (g / l): for gentamicin - 8, ciprofloxacin - 1, rifampicin - 0.125, doxycycline - 1.

In accordance with the developed evaluation criteria (see table 2), the resistance / sensitivity of the causative agent of tularemia to antibacterial drugs obtained indicators indicating that the studied strains are resistant to gentamicin and ciprofloxacin and sensitive to rifampicin and doxycycline (see table 6). A potentially infected employee is prescribed prophylactic treatment with rifampicin.

Example 3

Brucella abortus culture was isolated from beef, the source of human infection with brucellosis. The sensitivity of the selected culture to antibiotics of the 1st row was studied as described in Example 1. The results of DDM showed the following zones of growth inhibition: for gentamicin - 28 mm, doxycycline - 15 mm, ciprofloxacin - 42 mm and rifampicin - 20 mm. When using the serial dilution method in agar, the MPC of antibiotics was (g / l) for gentamicin - 1, ciprofloxacin - 1, rifampicin - 2, doxycycline - 8. In accordance with our assessment criteria scale (see table 4), the pathogen resistance / sensitivity brucellosis to antibacterial drugs received indicators (see table 7). The test culture is sensitive to gentamicin, ciprofloxacin and rifampicin, but resistant to doxycycline. The patient was prescribed treatment with gentamicin and rifampicin.

The use of the invention allows due to the developed scales of criteria for evaluating the antibiotic sensitivity of the strains Francusella tularensis and Brucella spp. implement as soon as possible (24-48 hours) and obtain reliable results for the selection of the most effective antibacterial drugs for the treatment of these especially dangerous infections.

Table 1 Antibacterial drugs Muller-Hinton Agar with Growth Additives pH 7.3 The value of the IPC, mg / l The diameter of the zones of growth suppression, mm Reference strains of F.tularensis Reference strains of F.tularensis subsp. tularensis schu subsp. holarctica 503 subsp. media-asiatica 543 subsp. tularensis schu subsp. holarctica 503 subsp. media-asiatica 543 Gentamicin one 2 one 32 25 thirty Amikacin 2 four 2 thirty 28 thirty Kanamycin 2 four 2 25 25 25 Streptomycin one 2 one thirty 25 thirty Doxycycline 0.5 one 0.5 thirty 25 25 Tetracycline 0.5 one 0.5 thirty 25 thirty Nalidixic acid one 2 one thirty 25 25 Ciprofloxacin 0.06 0.12 0.06 35 thirty 35 Ofloxacin 0.12 0.25 0.12 32 thirty 35 Pefloxacin 0.12 0.25 0.12 thirty thirty 35 Lomefloxacin 0.12 0.25 0.12 thirty 25 thirty Levofloxacin 0,03 0.06 0,03 35 thirty 35 Rifampicin 0.25 0.5 0.25 thirty 25 25

table 2 Antibacterial drugs The content of the drug in the disk (mcg) The diameter of the zones of inhibition of F.tularensis strains (mm) IPC values of F.tularensis strains (mg / L) Sensitive (S) Resistant (R) Sensitive (S) Resistant (R) Gentamicin 10 ≥25 ≤20 ≤4 ≥16 Amikacin thirty ≥25 ≤20 ≤8 ≥32 Kanamycin thirty ≥25 ≤20 ≤8 ≥32 Streptomycin thirty ≥25 ≤20 ≤4 ≥16 Doxycycline 10 ≥25 ≤20 ≤4 ≥16 Tetracycline thirty ≥25 ≤20 ≤4 ≥16 Nalidixic acid thirty ≥25 ≤20 ≤4 ≥16 Ciprofloxacin 5 ≥30 ≤25 ≤0.25 ≥1 Ofloxacin 5 ≥30 ≤25 ≤0.25 ≥1 Pefloxacin 10 ≥30 ≤25 ≤0.25 ≥1 Lomefloxacin 10 ≥25 ≤20 ≤1 ≥2 Levofloxacin 5 ≥30 ≤25 ≤0.25 ≥1 Rifampicin 5 ≥25 ≤20 ≤2 ≥8

Table 3 Antibacterial drugs Muller-Hinton Agar with Growth Additives pH 7.3 The value of the IPC, mg / l The diameter of the zones of growth suppression, mm Brucella reference strains Brucella reference strains B. abortus 544 B.melitensis 16 M B.suis 1330 B. abortus 544 B.melitensis 16 M B.suis 1330 Gentamicin 2 2 2 28 25 25 Amikacin four four four 28 25 25 Kanamycin four four four 25 25 25 Streptomycin 2 2 2 25 25 25 Doxycycline 2 0.5 one 25 thirty 25 Tetracycline one 0.5 one 25 thirty 25 Nalidixic acid 8 8 8 eighteen 19 19 Ciprofloxacin 0.5 0.5 0.5 thirty thirty thirty Ofloxacin one one 0.5 thirty thirty thirty Pefloxacin 2 four 2 28 thirty thirty Lomefloxacin 2 four 2 28 thirty thirty Levofloxacin 0.5 0.5 0.5 32 thirty thirty Rifampicin one 2 2 22 twenty twenty

Table 4 Antibacterial drugs The content of the drug in the disk (mcg) The diameter of the zones of inhibition of strains of Brucella (mm) MIC values of Brucella strains (mg / L) Sensitive (S) Resistant (R) Sensitive (S) Resistant (R) Gentamicin 10 ≥20 ≤15 ≤8 ≥32 Amikacin thirty ≥20 ≤15 ≤16 ≥64 Kanamycin thirty ≥20 ≤15 ≤16 ≥64 Streptomycin thirty ≥20 ≤15 ≤8 ≥32 Doxycycline 10 ≥20 ≤15 ≤8 ≥32 Tetracycline thirty ≥25 ≤20 ≤4 ≥16 Nalidixic acid thirty ≥15 ≤10 ≤32 ≥128 Ciprofloxacin 5 ≥30 ≤25 ≤4 ≥16 Ofloxacin 5 ≥25 ≤20 ≤8 ≥32 Pefloxacin 10 ≥25 ≤20 ≤8 ≥32 Lomefloxacin 10 ≥25 ≤20 ≤8 ≥32 Levofloxacin 5 ≥30 ≤25 ≤2 ≥4 Rifampicin 5 ≥15 ≤10 ≤8 ≥16

Claims (1)

A method for assessing the clinical effectiveness of antibacterial drugs for pathogens of especially dangerous infections Francisella tularensis and Brucella spp, including the cultivation of microbial cells in a nutrient medium using methods: disk diffusion and serial dilutions, characterized in that the assessment is carried out in three stages, according to the first stage, a criteria scale is developed antibiotic sensitivity for reference strains, while when creating a scale of criteria for Francisella tularensis using strains - F. tularensis subsp. nearctida., F. tularensis subsp. mediasiatica, F.tularensis subsp. holartia., when developing the criteria scale for Brucella spp., B. abortus, B.suis, B.melitensis are used; according to the second stage, the sensitivity of the test material to the same antibiotics is determined, and the third stage is to compare the latest indicators with the obtained scale for this type of microbe, while the first and second stages are carried out simultaneously using the same technology by plating a microbial suspension in a volume of 0.3 ml ( n · July ¹⁰ cfu / ml) for disc-diffusion method and the method of serial dilutions in nutrient medium with growth supplements, while as growth supplements Mueller-Hinton agar using magnesium sulfate - 0.05 g / l sulfate e iron - 0.1 g / l, L-cysteine - 0.6 g / l, lemon sodium - 1.2 g / l, potassium chloride - 2 g / l, potassium phosphate - 4 g / l, glucose - 15 g / l; further, the crops are incubated at 37 ° C for 24-48 hours and the results are recorded, and clinical efficacy is assessed by comparing the boundary values of the minimum inhibitory concentration and the diameters of the zones of inhibition of pathogen growth with the corresponding criteria scale, if the parameters of the studied material are included in the acceptable range of the criteria scale then the antibiotic is considered effective.