RU2481401C2 - METHOD TO DETECT β-LACTAMASE-PRODUCING OXIDASE-POSITIVE GRAM-NEGATIVE DIPLOCOCCI, SUSPICIOUS FOR APPURTENANCE TO MORAXELLA (BRANCHAMELLA) CATARRHALIS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method according to the invention discloses the possibility to detect β-lactamase-producing oxidase-positive gram-negative diplococci, suspicious for appurtenance to Moraxella (Branchamella) catarrhalis and provides for inoculation of a clinical material on blood-serum agar in sectors with simultaneous application of discs with penicillin 10 AU. The inoculation is incubated under regular atmospheric conditions for 18-24 hours, and in 36-48 hours from the moment of inoculation they assess availability or absence of a zone of growth delay for those microorganisms that are present in the clinical material. If there is no zone of microorganisms growth suppression around discs, they detect availability in the clinical material β-lactamase-producing oxidase-positive gram-negative diplococci. Appurtenance of detected diplococci to Moraxella (Branchamella) catarrhalis is identified by biochemical criteria.

EFFECT: simplified extraction of sought-for microorganisms from a contaminated clinical material.

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Description

The invention relates to medical microbiology and can be used for bacteriological diagnosis of infections caused by Moraxella (Branchamella) catarrhalis.

Moraxella (Branchamella) catarrhalis was first isolated from humans in 1896, until recently it was considered a normal inhabitant of the mucous membranes of the upper respiratory tract, but convincing data on its participation in the development of various infections have been accumulated over the past decades. M. catarrhalis causes purulent otitis media in 10-15% of cases, plays an important role in the occurrence of acute sinusitis, and takes the second or third place among bacterial pathogens of community-acquired pneumonia. Of the etiological agents in chronic bronchitis, M. catarrhalis is second only to Haemophilus influenzae and Streptococcus pneumoniae. 54.6% of all patients with M. catarrhalis infection are diagnosed with chronic obstructive pulmonary disease [3]. Until recently, it was believed that M. catarrhalis is usually highly sensitive to penicillin [5]. Currently, up to 95%, and according to some authors in almost 100% of cases, clinical isolates of M. catarrhalis produce beta-lactamase (test with nitrocefin) [1, 2, 7]. There are certain difficulties in identifying M. catarrhalis: 1) non-pigmented “saprophytic” neisseria, often contaminating sputum and normally living in the nasopharynx, have a similar morphology to M. catarrhalis — they grow in the form of pigmentless round opaque hemispherical whitish-gray colonies with a diameter of up to 2 3 mm; they do not form hemolysis, agar does not cause corrosion, bacterioscopy of cultures reveals gram-negative paired cocci, oxidase- and catalase-positive, since they belong to the same family of Neisseriaceae [3, 5, 6]; 2) in a culture study of the nasopharynx or sputum M. catarrhalis may not be identified due to growth inhibition by other predominant microorganisms: Neisseria sp., Streptococcus viridans, Corynebacterium sp., Staphylococcus sp., Streptococcus sp. and others. Normally located in the nasopharynx or contaminating sputum. Only the isolation of oxidase-positive, beta-lactam-positive, gram-negative diplococci allows us to suspect their belonging to M. catarrhalis.

Therefore, it is important to improve the laboratory diagnosis of inflammatory diseases of the respiratory system and ENT organs caused by M. catarrhalis.

The closest analogue is the definition of penicillin sensitivity described in Manual of clinical microbiology / editor in chief Patrick R. Murray. 7 ^th ed. - ASM PRESS Washington, 1999, 1773 p. on page 544. According to it, a pure culture of oxide-positive, indolenegative, asacaralitic cocco-like form of non-enzyme is seeded on a surface of blood agar with 5% mutton blood and then immediately apply a 10 DB penicillin disk. and incubated at 35 ° C under normal atmospheric conditions for 18-24 hours. Any strain with a zone of growth inhibition around the penicillin disk is interpreted as sensitive for identification purposes.

The aim of our proposed method is to facilitate the isolation of β-lactamase-producing oxidase-positive gram-negative diplococci suspected of belonging to Moraxella (Branchamella) catarrhalis from contaminated clinical material (naturally coughing up or induced sputum, transtracheal aspirate) or from a clinical microorganism normal pharynx, nasopharynx).

The method provides the following. On blood serum agar in Petri dishes with a diameter of 90 or 100 mm per 1/2 cup area (first sector), the clinical material (nasal swabs, pharynx, nasopharynx, naturally coughing or induced sputum, transtracheal aspirate) is seeded with a continuous lawn, followed by sieving two more sectors to obtain isolated colonies of microorganisms. Then, a penicillin disk (10 units) is placed on the surface of the first sector. The plates are incubated at 37 ° C. under normal atmospheric conditions for 18-24 hours. A high concentration of penicillin inhibits the growth of most other microorganisms that are representatives of the normal microflora of the respiratory tract (neusseries, staphylococci, streptococci, etc.), which makes it possible to obtain around β-lactamase-producing oxidase-positive gram-negative diplococci suspicious around the penicillin disk catarrhalis from clinical material. Subsequently, biochemical identification of this culture is performed.

The essence of the method:

1. On blood-serum agar in Petri dishes with a diameter of 90 or 100 mm per 1/2 cup area (first sector), the clinical material is sown with a continuous lawn, followed by sieving into two more sectors to obtain isolated colonies of microorganisms.

2. Then put a penicillin disk (10 units) on the surface of the first sector.

3. Incubate plates at 37 ° C under normal atmospheric conditions for 18-24 hours.

4. To assess the presence or absence of a zone of growth inhibition of microorganisms around the disk with penicillin 10 PIECES.

Known methods for determining the sensitivity to penicillin in M. catarrhalis, which are used to identify this microorganism and for its differentiation from other representatives of the genus Moraxella [6, 8]. For this test, a pure daily culture of a strain suspected of belonging to M. catarrhalis is required, that is, 36-48 hours should elapse from the moment the clinical material is inoculated to the test results.

In common with the prototype: 1) use of a disk with penicillin 10 PIECES; 2) applying it immediately after sowing; 3) incubation of inoculation under normal atmospheric conditions for 18-24 hours.

Distinctive features of the prototype: 1) not a pure culture of the microorganism strain is applied, but the clinical material itself; 2) sowing is performed on blood serum agar; 3) incubation is carried out at 37 ° C; 4) the inoculation of clinical material is done by sectors to obtain isolated colonies of microorganisms.

In our proposed method, it is possible to suspect a culture of belonging to M. catarrhalis producing β-lactamase on the first day after sowing clinical material. Therefore, the final identification and delivery of the result of antibiotic sensitivity is possible on the second day from the moment of seeding of clinical material.

The authors conducted studies to identify β-lactamase-producing oxidase-positive gram-negative diplococci suspected of belonging to M. catarrhalis from clinical material (pharynx, nose, sputum) using this method. In 2007, 19 strains of M. catarrhalis were isolated: 2 strains from sputum (in children with the following pathology: chronic obstructive pulmonary disease, pneumonia); 1 strain isolated from pharynx (exacerbation of chronic tonsillitis); 16 strains isolated from the nose (chronic rhinosinusitis, sinusitis, ethmoiditis, mastoiditis, subtrophic rhinitis). As a result: around the penicillin disk (10 PIECES) there is no growth suppression zone in β-lactamase-producing oxidase-positive gram-negative diplococci suspected of belonging to M. catarrhalis, which were subsequently identified by biochemical characteristics as M. catarrhalis. In contrast to those identified as S. viridans, S. pneumoniae, Streptococcus pyogenes, Neisseria sp., Staphylococcus sp., Which form a growth suppression zone around the penicillin disk (10 PIECES), with an average diameter of 20 mm.

The authors also tested museum test strains by sowing the daily culture of each individually with a continuous lawn on blood serum agar and then applying a penicillin disk (10 units) on the agar surface. The plates were incubated at 37 ° C under normal conditions for 18-24 hours. The results are presented in table 1.

Table 1 Testing of museum test strains for penicillin (disk 10 UNITS.) Museum Test Strain Diameter of growth suppression zone in millimeters Streptococcus pneumoniae ATCC 49619 25 Staphylococcus aureus 25923, NCDC, USA thirty Staphylococcus haemolyticus * producing β-lactamase 0 Streptococcus pyogenes * thirty Streptococcus agalactiae * 25 Neisseria meningitides * 25 Moraxella catarrhalis * producing β-lactamase 0 Moraxella catarrhalis * not producing β-lactamase 25 Corynebacterium diphtheriae mitis toxigenes 090086 33 Corynebacterium diphtheriae gravis 665 36 Corynebacterium ulcerans * 35 * - from the working museum - cultures isolated from clinical material and used to control bacteriological culture media with typical properties corresponding to microorganisms of recognized national and international collections [4].

References:

1. Avdeev S.N., Chuchalin A.G. The role of bacterial infection and the choice of antibiotics in exacerbation of chronic bronchitis / S.N. Avdeev, A.G. Chuchalin // Consilium Medicum. - 2000. - T.2, No. 10. - S. 5-9.

2. Zubkov M.N. Modern aspects of the etiological diagnosis and antimicrobial therapy of community-acquired pneumonia / MN Zubkov // Farmateka. - 2005. - No. 19. - S.31-37.

3. Zubkov M.N. Moraxella (Branchamella) catarrhalis: role in human pathology, identification and antibiotic resistance / MN Zubkov // Infection and antimicrobial therapy. - 2001. - T.3, No. 4. - S.115-116.

4. MUK 4.2.2316-08 "Methods for the control of bacteriological culture media."

5. The determinant of bacteria Bergey. In 2 vols. T.1: Trans. from English Ed. J.Houlta, N.Krieg, P. Snit, J. Staley, S. Williams. - M .: Mir, 1997 .-- 432 p.

6. Key to non-trivial pathogenic gram-negative bacteria (aerobic and optionally anaerobic). Per. from English R. Weyant, W. Moss, R. Weaver, D. Hollis, J. Jordan, E. Cook, M. Deinshwar. - M .: Mir, 1999 .-- 791 p.

7. Strachunsky L.S., Kamanin E.I. Antibacterial therapy of infections in otorhinolaryngology / L.S. Strachunsky, E.I.Kamanin // Russian Medical Journal. - 1998. - T.6, No. 17. - S.684-693.

8. Manual of clinical microbiology / editor in chief Patrick R. Murray. 7 ^th ed. - ASM PRESS Washington, 1999, 1773 p.

Claims (1)

A method for detecting β-lactamase-producing oxidase-positive gram-negative diplococci suspected of belonging to Moraxella (Branchamella) catarrhalis, characterized in that the clinical material is plated on blood-serum agar with sectors to obtain isolated colonies and at the same time, 10 units of penicillin are placed on the plating, incubated with 10 units of penicillin. within 18-24 hours at a temperature of 37 ° C under normal atmospheric conditions, after 36-48 hours from the time of sowing, the presence or absence of a zone of growth inhibition of microorganisms is assessed, The operator occurring in clinical material, and in the absence of growth inhibition zone around the disk to detect the presence of clinical material β-lactamase oksidazopozitivnyh gram negative diplococci and belonging to the identified diplococci Moraxella (Branchamella) catarrhalis were identified by biochemical characteristics.