Method of increasing sensitivity of microorganisms to antimicrobial agents

FIELD: biotechnology.

SUBSTANCE: two suspensions are prepared. Clinical polyantibiotic-resistant strains Escherichia coli are added to an isotonic solution of NaCl to achieve the concentration of 30-40 thousand CFU/ml. Copper nanoparticles are added to the solution of NaCl to achieve the concentration of 0.01-0.05 mg/ml. The suspension of ethylenediaminetetraacetic acid - EDTA is prepared by its dilution in distilled water at the rate of 0.1-0.2:1, respectively. NaOH is added to the prepared suspension to obtain the solution of EDTA with pH=7.6-8. The prepared suspensions are connected with the solution of EDTA in the following ratios by wt %: suspension of copper nanoparticles - 70-85, suspension of microorganisms - 10-20, EDTA - 5.10. It is incubated in the shaker at 100-150 rev/min and a temperature of 36-38°C for 40-60 minutes. The resulting biomass is inoculated on the solid nutrient medium with the volume of 20-25 ml in the amount of 0.1-0.12 ml. It is incubated in the thermostat at a temperature of 36-38°C for 18-24 hours. The sensitivity of E.coli strains to antibiotics is determined.

EFFECT: invention enables to increase the sensitivity of the said bacterial strains to antibiotics gentamicin and ampicillin.

2 tbl, 2 cl, 1 ex

 

The invention relates to medicine, namely to Microbiology. This solution can be used to overcome antibiotic resistance of microorganisms and increase the effectiveness of antibiotic therapy.

Despite continuous high flow and development of new antibacterial drugs, the problem of infectious-inflammatory diseases remains one of the most complex and urgent problems for doctors all over the world. The use of antibiotics in medicine contributes to the emergence and spread of drug resistance [paly G.K., paly I.G. Characterization of antibiotic susceptibility of pathogens of infectious and inflammatory diseases // website: http://www.provisor.com.ua/archive/2000/N12/antibact.php].

Known methods of increasing the sensitivity of microorganisms to antibiotics by exposure to various tools and chemicals, proteolytic bacterial enzymes, enzymes of animal origin. Some of these tools are different mineral water [patent RU invention No. 2207863, 2230563, 2246541, 2255746, application EN for the invention №2003103843].

However, the increased sensitivity when using these funds to be insignificant and has no statistical validity and does not reach the level when using disco is diffuzionnogo method allows to consider the strain is sensitive to this antibiotic. A change in the quantitative indicators of the sensitivity of the strains vary within the error.

Also known "Method of increasing the sensitivity of microorganisms to antibiotics" [RU patent for the invention №2052198], based on the effects of bacterial origin. As the active substance, while providing antibacterial, anti-inflammatory and immunostimulatory effects, using an aqueous solution of Palestina. In vitro use of elastin at a final concentration of 0.1-0.4%, while carry out the incubation of microorganisms with Palastina within three days. Elastin local use within three days at a concentration of 0.01 to 0.04%.

However, the use of bilastine can lead to the development of allergic reactions. This drug is contraindicated in hypersensitivity and violations of the integrity of the skin, which complicates its use in the local treatment of wound infection (instructions for use; http://healthoffice.ru/content/pilastin). In addition, the application of this method requires a long time of exposure to the drug, namely in the course of 3 days, which lengthens the time frame for achieving the desired effect.

There is also known a method of increasing the antibiotic sensitivity of microorganisms [RU patent for the invention №2053773], which consists in carrying out sequential or simultaneously the military with antibiotic treatment antibiotic resistant strains of microorganisms 2.5-5 mg/l solutions of silver, obtained by an electrolytic method and brought to isotonicity by the addition of sodium chloride.

However, this method involves the use as raw materials of silver, which is a more expensive material than copper. In addition, this method involves the joint action of silver and antibiotics, which is not always effective in the treatment of wound infections caused by antibiotic-resistant strains of microorganisms, as in clinical practice requires different methods of introduction of antibacterial agents (parenteral, oral etc), not just local.

The closest analogue to the claimed invention is a method of increasing the sensitivity of microorganisms to antibiotics [study of the influence of metal nanoparticles on sensitivity to antibiotics of clinical strains of microorganisms / I.V. Babushkin [and other]//Bulletin of new medical technologies. 2011. T. XVIII, No. 2. S-513], is the impact on microorganisms suspension of nanoparticles of copper, iron, zinc and their alloy at a concentration of 0.01 mg/ml for 30 minutes.

However, the proposed method is effective against strains of Pseudomonas aeruginosa and involves the restoration of sensitivity to only one of the antibiotics group cheapaspirin - ceftazidime.

Task claimed the image is to be placed is providing a significant increase in the quantities of antibiotic susceptibility of microorganisms and the possibility of increasing the sensitivity is widely used in the world cheap antibiotics in particular, gentamicin and ampicillin.

The essence of the invention is characterized by the fact that they are preparing a two-suspended by adding isotonic NaCl solution: copper nanoparticles to achieve a concentration of 0.01-0.05 mg/ml and microorganisms to achieve their concentration 30-40 thousand CFU/ml; prepare a suspension of ethylenediaminetetraacetic acid EDTA by dilution in distilled water at the rate of 0.1 to 0.2:1, respectively, added to this suspension NaOH to obtain EDTA with pH=7,6-8; connect the prepared suspension and EDTA in the following ratios, wt%:

a suspension of nanoparticles of copper - 70-85,

a suspension of microorganisms - 10-20,

the EDTA - 5-10,

and incubated in a shaker at 100 to 150 rpm and a temperature of 36-38°C for 40-60 minutes; sow obtained biomass on solid nutrient medium with a volume of 20-25 ml in the amount of 0,1-0,12 ml and incubated in a thermostat at a temperature of 36-38°C for 18-24 hours, at the same time as the microorganisms use clinical polyetheretherketone strains of Escherichia coli (E. coli).

Applying the method with the above-described characteristics, in which the solid nutrient medium used mastopathy agar.

The technical result of the claimed invention.

The above objective is achieved by using the method defined compo is having their percentages and developed by the authors of the technical parameters to be taken when implementing this method of manipulation. Copper nanoparticles provide for the elimination of extrachromosomal genetic material - plasmid DNA carrying genes for antibiotic resistance. Thus, the use of copper nanoparticles allows you to completely destroy the plasmid DNA, which provides a significant increase in the quantitative values of sensitivity indicated earlier antibiotics - gentamycin and ampicillin. The latter is widely applicable in the world, cheap and at this point of time there is a large percentage of microorganisms with resistance to them. The use of EDTA increases membrane permeability of bacterial cells to nanoparticles of metals, facilitating the penetration of copper nanoparticles to the genetic material of the bacterial cell wall and changes in it that enhance the antibiotic sensitivity of strains.

Method of increasing the sensitivity of microorganisms to antimicrobial agents is as follows. Prepare a suspension. To do this, add in isotonic NaCl solution in separate containers copper nanoparticles to achieve their concentration in the solution is 0.01-0.05 mg/ml and microorganisms - clinical polyetheretherketone strains of E. coli to the pros who supply their concentration 30-40 thousand CFU/ml Prepare a suspension of EDTA by dilution in distilled water at the rate of 0.1 to 0.2:1, respectively. Under the control of the pH-meter added to the resulting suspension of NaOH to obtain EDTA with pH=7,6-8 with constant stirring. Then connect the prepared suspension and EDTA in the following proportions: 70-85% suspension of nanoparticles of copper, 10-20% suspension of microorganisms and 5-10% solution of EDTA. The resulting mixture is incubated at a temperature of 36-38°C and 100-150 rpm for 40-60 minutes. Sow obtained biomass on solid nutrient medium with a volume of 20-25 ml in the amount of 0,1-0,12 ml and incubated in a thermostat at a temperature of 36-38°C for 18-24 hours. In most cases, as a solid nutrient medium used mastopathy agar.

Example.

Allocated from patients with purulent complications and orthopaedic profile 10 clinical strains of E. coli with resistance to ampicillin and gentamicin. Strains of microorganisms were cultured on mesopatamia agar in an incubator for 24 hours at 37°C. Then prepared 10 tubes containing 0,85 ml suspension of copper nanoparticles with a concentration of 0.01 mg/ml in isotonic NaCl; 10 tubes containing 0,85 ml isotonic NaCl; 20 tubes containing 0.1 ml of suspension previously isolated microorganisms with a concentration of 30 thousand CFU/ml from onicescu NaCl two for each strain. Also prepared by the above method 1 ml of EDTA with pH=8. Combined contents of the test tubes so that received 10 tubes with the mixture of the suspension of each microorganism with a suspension of nanoparticles of copper (experimental group) and 10 tubes with the mixture of the suspension of each microorganism with isotonic (group comparison). Added to each of the 20 received tubes of 0.05 ml of a pre-prepared solution of EDTA. Incubated the mixture in a shaker for 30 minutes at 100 rpm and 37°C. After were sown in individual cups with mesopartner agar in 0.1 ml of each of the received biomass, and incubated at 37°C for 24 hours. At the end of the incubation period produced a sensitivity study has grown strains of microorganisms to antimicrobial drugs gentamycin, ampicillin disco-diffusion method in accordance with ANGUISH 4.2.1890-04 "Determination of the sensitivity of microorganisms to antibiotics".

As a result of the research revealed the following.

Antibiotikoustoichivosti clinical strains of E. coli were:

AntibioticsThe diameter of zone of growth inhibition of E. coli, mm
Gentamicin 6,2±1,3
Ampicillin9,0±1,7

Dynamics of change of antibiotic susceptibility of clinical strains of Escherichia coli:

AntibioticsThe diameter of zone of growth inhibition of E. coli, mm
Incubation with isotonicIncubation with suspension of nanoparticles of copper and EDTA
Gentamicin6,7±1,415,1±1,1*
Ampicillin9,1±1,418,1±0,5*
Note: *p<0,001, where p is the confidence level of the differences in performance compared to the comparison group.

In connection with the above quantitative parameters, it was observed the following. In microorganisms in the comparison group was not statistically significant changes in the zone of growth inhibition against ampicillin and gentamicin. The microorganisms of the experimental group was statistically significant increase of the zone of growth inhibition against the studied antibiotics. In accordance with ANGUISH 4.2.1890-04 "determination of the sensitivity of microor is Anisimov to antibacterial preparations of antibiotic-resistant clinical strains of E. coli in the experimental group significantly increased the sensitivity to antibiotics, particularly to gentamicin and ampicillin, which are the drugs of choice for treatment of infections caused by strains of E. coli.

1. Method of increasing the sensitivity of microorganisms to antimicrobial agents, characterized by the fact that preparing two suspension by adding isotonic NaCl solution: copper nanoparticles to achieve a concentration of 0.01-0.05 mg/ml and microorganisms to achieve their concentration 30-40 thousand CFU/ml; prepare a suspension of ethylenediaminetetraacetic acid EDTA by dilution in distilled water at the rate of 0.1 to 0.2:1, respectively, added to this suspension NaOH to obtain EDTA with pH=7,6-8; connect the prepared suspension and EDTA in the following ratios, wt%:
a suspension of nanoparticles of copper - 70-85,
a suspension of microorganisms - 10-20,
the EDTA - 5-10,
and incubated in a shaker at 100 to 150 rpm and a temperature of 36-38°C for 40-60 min; sow obtained biomass on solid nutrient medium with a volume of 20-25 ml in the amount of 0,1-0,12 ml and incubated in a thermostat at a temperature of 36-38°C for 18-24 hours, at the same time as the microorganisms use clinical polyetheretherketone strains of Escherichia coli.

2. The method according to claim 1, characterized in that as a solid nutrient medium used meats is peptone agar.



 

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