RU2794355C1 - Method for primary plating of periodontal pockets - Google Patents

Abstract

FIELD: medicine; microbiology; dentistry.

SUBSTANCE: invention relates to a method for primary plating of periodontal pockets.

EFFECT: invention allows to improve the diagnosis of infectious and inflammatory periodontal diseases of bacterial aetiology by obtaining the maximum number of types of clinically significant microorganisms separated from periodontal pockets from patients with periodontitis in the primary culture.

1 cl, 2 ex

Description

The field of technology to which the invention belongs

SUBSTANCE: invention relates to medicine, namely to microbiology and dentistry, and can be used for primary inoculation of biomaterial isolated from the oral cavity of patients with periodontal diseases during a dental examination.

State of the art

A known method for rapid diagnosis of Escherichia coli and bacteria of the Escherichia coli group in the oral cavity, carried out as follows: the patient is taken biological material from the oral cavity: oral and gingival fluids using a micropipette, smear-imprint from the oral mucosa - with a sterile cotton swab, dental biofilm using an excavator (Ekradent, Russia). The biomaterials are placed in the Koda nutrient medium (reg. certificate RZN 2013/699, TU 20.59.52-222-19862939-2018), poured into Eppendorf-type microtubes, in a volume of 1.8 ml. The microtubes are incubated in a thermostat TC-80 (Ankar, Russia) for 24 hours at 37°C. After that, the change in color and turbidity of the nutrient medium of the Code is evaluated. If the original green color and transparency of the medium are preserved, E. coli and BGKP are absent in the sample. The color change to intense yellow with the formation of gas bubbles and turbidity of the medium in the sample indicate the presence of E. coli and BGKP [RF Patent for invention No. 2732412, Application 2020108748, 27.02.2020, Published: 16.09.2020 Bull. No. 26].

The disadvantages of this method are the low sensitivity and specificity of the method, as well as the lack of conditions for the cultivation of microflora specific to the oral cavity, including anaerobic microorganisms.

There is a known method of cultivating bacteria of the genus Leptotrichia - residents of the microflora of the oral cavity, carried out as follows: after taking clinical material from the patient's oral cavity in the dentist's office, it is placed in a transport medium for anaerobes and delivered to the bacteriological laboratory within 1-2 hours. Dilutions of 10 ^-3 -10 ^-5 are made from the obtained material with a sterile 0.85% NaCl solution. The primary inoculation of the material from the prepared dilutions is carried out with a calibrated bacteriological loop (0.2 mm, capacity 0.005 ml) on the surface of the Uriselect plate medium by the method of sector inoculation into four sectors, each time firing the loop, into 90 mm Petri dishes and then the primary inoculations are incubated in anaerobic and microaerophilic (5%-10% CO ₂ ) conditions, t _opt =+35+37°C for 48-72 hours. In the absence of growth, the cups were repeatedly kept for another 24 hours to exclude false negative results caused by insufficient incubation [RF Patent for invention No. 2441908, Application 2010110394/10, 03/18/2010, Published: 02/10/2012 Bull. No. 4].

The disadvantages of this method are the lack of nutrient media for the cultivation of anaerobes in the proposed scheme, which limits the possibility of their isolation from the biomaterial.

There is a known method for predicting negative consequences in the oral cavity during orthodontic treatment of dentoalveolar anomalies with fixed equipment, carried out as follows: the patient's dental plaque is taken before brushing the teeth and placed in a thioglycol nutrient transport medium for subsequent transportation. A series of two-fold dilutions of the starting material 10 ^-2 -10 ^-12 is prepared for inoculation of microorganisms on appropriate nutrient media on 5% blood agar with sodium azide, on 5% blood hemin agar to detect streptococci. Cultivation is carried out at a temperature of 37°C. After temperature control, a quantitative count of the colonies of each species is carried out, taking into account the inoculation dose and the degree of dilution of the biosubstrate. Species identification of isolated pure cultures of microorganisms is carried out on the basis of the study of their morphological, tinctorial, cultural, biochemical and antigenic properties in accordance with the determinant of Bergy (J. Holt, 1997). The quantitative and qualitative characteristics of streptococci of the oral cavity are assessed. The quantitative content of oral streptococci S. mutans and S. sanguis is determined by the bacteriological method of microbiological research, the concentration is expressed through the decimal logarithm of the size of the grown colonies (lg CFU / ml). The interpretation of the obtained data is carried out in accordance with the guidelines for bacteriological diagnostics (RF industry standard OST 91500.11.0004-2003 "Protocol for managing patients. Intestinal dysbacteriosis") [RF Patent for invention No. 2639476, Application 2014124674, 17.06.2014, Published: 21.12. 2017 Bull. No. 36].

The disadvantages of this method are the lack of nutrient media for the cultivation of anaerobes in the proposed scheme, which limits the possibility of their isolation from the biomaterial.

The prototype is the method of primary seeding of biomaterial isolated from the oral cavity of patients with cystic fibrosis during a dental examination. The essence of the method is as follows: on the surface of two plates with 5% blood agar with defibrinated sheep blood, two plates with universal chromogenic medium, one plate each with selective medium for Burkholderia cepacia (OFPBL-agar) with bacitracin and polymyxin sulfate, Veilonella-agar , anaerobic agar, Clostridium agar, lactobacilli agar, bifidobacteria agar, biomaterial is inoculated from the oral cavity of patients with cystic fibrosis, taken during a dental examination, using the "stroke" technique; on the surface of the plates with Sabouraud agar with chloramphenicol, the homogenized material is inoculated by the plaque method, followed by incubation at 28°C for up to 14 days with daily views of the crops; then one inoculated plate with 5% blood agar with defibrinated sheep blood and with universal chromogenic medium are incubated in a thermostat at a temperature of 37°C for 24-48 hours with daily viewing of crops; dishes with selective medium for Burkholderia cepacia (OFPBL-agar) with bacitracin and polymyxin sulfate are incubated under aerobic conditions for 24-48 hours at a temperature of 37°C, then incubated for up to 14 days at a temperature of 28°C with daily viewing of crops; one inoculated plate each with 5% blood agar with defibrinated sheep blood and universal chromogenic medium, as well as plates with Veilonella agar, anaerobic agar, Clostridium agar, lactobacillus agar, bifidobacteria agar are incubated under anaerobic conditions for 120 hours at a temperature 37 ° C with a review after the end of the incubation period [RF Patent for invention No. 2779641, Application 2022105520, 03/01/2022, Published: 09/12/2022 Bull. No. 26].

The disadvantages of this method are inoculation by the "stroke" method, which eliminates the isolation of microorganisms in titers less than 10 ³ and also makes it difficult to isolate microorganisms in the case of a large microbial load in the biological material; the set of proposed media is not provided for isolating a large species diversity of obligate anaerobic microorganisms that have the greatest clinical significance in periodontopathogenic processes.

Disclosure of invention

The objective of the invention is to improve the diagnosis of infectious and inflammatory periodontal diseases of bacterial etiology by obtaining the maximum number of types of clinically significant microorganisms separated from the oral cavity from patients with periodontitis in the primary seeding.

This is achieved by the fact that, unlike the known methods of primary seeding discharged from the oral cavity, a disposable dental probe is used to take the biomaterial from the periodontal pocket of a patient with periodontitis. Next, a disposable probe with biomaterial is placed in a test tube with 2 ml of thioglycol nutrient transport medium, which allows maintaining the viability of aerobic and anaerobic microorganisms and is delivered to the bacteriological laboratory within 2 hours, where seeding is carried out on 7 nutrient media: 5% blood agar with defibrinated sheep blood [Atlas, Ronald M., 1946-. Handbook of microbiological media. - Taylor & Francis, 2010-01-01.], universal chromogenic medium [1. Pezzlo M (1998), Clinical Microbiology Reviews 1:268-280; 2. Wilkie M. E., Almond M. K., Marsh F. P. (1992), British Medical Journal 305:1137-1141; 3 Friedman M.P. et al (1991), Journal of Clinical Microbiology, 29:2385-2389; 4. Murray P., Traynor P. Hopson D., (1992), Journal of Clinical Microbiology 30:1600-1601; 5. Soriano F., Ponte C., (1992), Journal of Clinical Microbiology 30:3033-3034; 6 Merlino et al (1995) Abstr. Austr. microbiol. 16(4):17-3.], Veilonella agar [Registration certificate for a medical device FSS 2009/03709], anaerobic agar [Registration certificate for a medical device FSS 2009/03707], Clostridium agar [Registration certificate for a medical device FSZ 2009/03707], agar for lactobacilli [Registration certificate for a medical product FSZ 2009/03709], Brucella agar with the addition of 7% defibrinated sheep blood [Registration certificate for a medical device FSZ 2009/03709]. Additionally, disks with antibacterial drugs are placed on Petri dishes with nutrient media: ampicillin 10 µg, ciprofloxacin 5 µg, erythromycin 15 µg; then seeded cups with disks with antibacterial drugs are incubated under anaerobic conditions for 120 hours at a temperature of 37°C.

The technical result of the invention lies in the fact that the claimed method of primary seeding allows you to simultaneously select the maximum number of microorganisms of clinical importance in the development of dental diseases, in particular caries and periodontal diseases: bacteria of the genera Capnocytophaga spp., Eikenella spp., Veilonella spp., Actinomyces spp. ., Aggregatibacter spp, Streptococcus spp., Porphyromonas spp., Campylobacter spp., Fusobacterium spp., Eubacterium spp., Prevotella spp., most of which are difficult to cultivate obligate anaerobes.

Implementation of the invention

The essence of the proposed method is as follows: a biomaterial is taken from the periodontal pocket of a patient with periodontitis with a disposable dental probe. Next, a disposable probe with biomaterial is placed in a test tube with 2 ml of thioglycol nutrient transport medium, which allows maintaining the viability of aerobic and anaerobic microorganisms and is delivered to the bacteriological laboratory within 2 hours. In the bacteriological laboratory, the selected material is vortexed, 50 µl of thioglycol nutrient medium is taken and inoculated on the surface of Petri dishes with all types of nutrient media: 5% blood agar with defibrinated sheep blood, universal chromogenic medium, Veilonella agar, anaerobic agar, Clostridium agar , lactobacillus agar, Brucella agar with 7% defibrinated sheep blood using the "lawn" technique. Then disks with antibacterial preparations are placed on the surface of nutrient media: ampicillin 10 µg, ciprofloxacin 5 µg, erythromycin 15 µg; then seeded cups with disks with antibacterial drugs are incubated under anaerobic conditions for 120 hours at a temperature of 37°C.

In the proposed method for the primary inoculation of biomaterial isolated from the oral cavity of patients with periodontal diseases during a dental examination, optimal conditions are provided for the isolation of clinically significant microorganisms: a set of 7 nutrient media, incubation under anaerobic conditions make it possible to identify the maximum number of species of unpretentious and difficult to cultivate microorganisms; the addition of 3 antibacterial disks when cultivating inoculated plates under anaerobic conditions inhibits the growth of normal microflora of the oral mucosa, which can suppress the isolation of clinically significant bacteria.

The implementation of the proposed method can be demonstrated by the following examples:

Example 1

Patient N., 45 years old. Diagnosis: chronic periodontitis. The laboratory received the material obtained during the dental examination - detachable periodontal pocket. Produced primary sowing in two ways: the method described in the prototype, as well as our proposed method. The following results were obtained:

The sowing method specified in the prototype Our proposed method 5% defibrinated sheep blood agar Streptococcus oralis 10 ⁴
Streptococcus parasanguinis 10 ³ Streptococcus oralis 10 ⁴
Streptococcus mitis 10 ⁴
Streptococcus constellatus 10 ⁴
Aggregatibacter aphrophilus 10 ⁴ Universal chromogenic medium Streptococcus oralis 10 ⁵
Streptococcus mitis 10 ⁴
Streptococcus parasanguinis 10 ³ Streptococcus oralis 10 ⁴
Streptococcus mitis 10 ³
Streptococcus parasanguinis 10 ⁴
Streptococcus constellatus 10 ⁴ Veilonella agar Neisseria perflava 10 ³
Veilonella parvula 10 ⁴ Neisseria perflava 10 ³
Veilonella parvula 10 ⁴
Lactobacillus salivarius 10 ⁴ Agar for anaerobes Streptococcus anginosus 10 ³
Streptococcus oralis 10 ³
Actinomyces odontolyticus 10 ³ Capnocytophaga ochracea 10 ⁴
Eikenella corrodens 10 ⁴
Streptococcus anginosus 10 ⁴
Streptococcus oralis 10 ⁴
Actinomyces odontolyticus 10 ³ Clostridium agar Streptococcus anginosus 10 ⁴
Streptococcus constellatus 10 ⁴ Streptococcus anginosus 10 ⁴
Capnocytophaga ochracea 10 ⁴
Streptococcus constellatus 10 ³
Actinomyces odontolyticus 10 ⁴ Agar for lactobacilli Lactobacillus salivarius 10 ⁴ Streptococcus gordonii 10 ⁴
Eikenella corrodens 10 ⁴
Lactobacillus salivarius 10 ⁴ - Brucella agar with 7% defibrinated sheep blood - Eikenella corrodens 10 ⁵
Aggregatibacter aphrophilus 10 ⁵
Leptotrichia hofstadii 10 ⁴
Prevotella intermedia 10 ⁴

Thus, thanks to the application of our proposed method, the growth of 15 types of microorganisms on nutrient media was obtained, while using the method specified in the prototype, the growth of 10 types of microorganisms was obtained. When using our method of sowing, the growth of clinically significant anaerobic paradontopathogenic flora was obtained - Aggregatibacter aphrophilus, Capnocytophaga ochracea, Prevotella intermedia, Streptococcus gordonii, which would be impossible to obtain using only the list of nutrient media specified in the prototype, and also without the use of disks with antibacterial drugs due to the metabolic characteristics of these microorganisms.

Example 2:

Patient T., 54 years old. Diagnosis: chronic periodontitis. The laboratory received the material obtained during the dental examination - detachable periodontal pocket. Produced primary sowing in two ways: the method described in the prototype, as well as our proposed method. The following results were obtained:

The sowing method specified in the prototype Our proposed method 5% defibrinated sheep blood agar Streptococcus vestibularis 10 ⁵
Staphylococcus aureus 10 ⁴ Streptococcus constellatus 10 ⁴
Rothia dentocariosa 10 ⁵ Streptococcus vestibularis 10 ⁵
Staphylococcus aureus 10 ³
Streptococcus constellatus 10 ⁴
Rothia dentocariosa 10 ⁵
Fusobacterium nucleatum 10 ³
Porphyromonas gingivalis 10 ³ Universal chromogenic medium Streptococcus vestibularis 10 ⁴
Staphylococcus aureus 10 ³
Rothia dentocariosa 10 ⁴ Streptococcus vestibularis 10 ⁵
Staphylococcus aureus 10 ³
Rothia dentocariosa 10 ⁵ Veilonella agar Veilonella parvula 10 ⁴ Veilonella parvula 10 ⁴
Veilonella atypica 10 ³ Agar for anaerobes Actinomyces odontolyticus 10 ³
Streptococcus constellatus 10 ⁴ Actinomyces odontolyticus 10 ³
Streptococcus constellatus 10 ⁴
Actinomyces oris 10 ³ Clostridium agar Actinomyces odontolyticus 10 ³
Streptococcus constellatus 10 ³ Actinomyces odontolyticus 10 ³
Streptococcus constellatus 10 ⁴ Agar for lactobacilli Lactobacillus salivarius 10 ⁴
Lactobacillus fermentum 10 ³ Lactobacillus salivarius 10 ³
Lactobacillus fermentum 10 ⁴ - Brucella agar with 7% defibrinated sheep blood - Actinomyces oris 10 ⁴
Actinomyces odontolyticus 10 ⁴
Aggregatibacter aphorophilis 10 ³
Streptococcus intermedius 10 ⁵
Fusobacterium nucleatum 10 ⁴
Porphyromonas gingivalis 10 ³

Thus, thanks to the use of our proposed method, the growth of 14 types of microorganisms on nutrient media was obtained, while using the method specified in the prototype, the growth of 10 types of microorganisms was obtained. When using our method of seeding, the growth of clinically significant anaerobic paradontopathogenic flora was obtained - Veilonella atypica, Aggregatibacter aphrophilus, Fusobacterium nucleatum, Streptococcus intermedius, Porphyromonas gingivalis, which would be impossible to obtain using only the list of nutrient media specified in the prototype, and also without the use of disks with antibacterial drugs due to the metabolic characteristics of these microorganisms.

The proposed method allows to evaluate the qualitative and quantitative composition of the microflora of the mucous membranes of the oral cavity, to identify and differentiate paradontopathogenic bacteria, which helps to optimize the therapy of patients with periodontal tissue diseases.

Claims (1)

A method for primary seeding of discharge from periodontal pockets, including taking biomaterial from the periodontal pocket of a patient with periodontitis, characterized in that a disposable probe with biomaterial is placed in a test tube with 2 ml of thioglycol nutrient transport medium and delivered to a bacteriological laboratory within 2 hours, where, after vortexing, inoculation of material from thioglycol medium in a volume of 50 µl for each of 7 nutrient media: 5% blood agar with defibrinated sheep blood, universal chromogenic medium, Veilonella agar, anaerobic agar, Clostridium agar, lactobacillus agar, Brucella agar with adding 7% defibrinated sheep blood; after sowing, disks with antibacterial drugs are placed on the surface of nutrient media: ampicillin 10 µg, ciprofloxacin 5 µg, erythromycin 15 µg; then seeded cups with disks with antibacterial drugs are incubated under anaerobic conditions for 120 hours at a temperature of 37°C.