A novel and special solid growth medium for susceptibility testing of colistin by the diffusion method

Abstract

The invention relates to a novel and specialised solid growth medium for use in susceptibility testing of polymyxins, such as colistin, using the diffusion method.

Description

[0001] A NOVEL AND SPECIAL SOLID GROWTH MEDIUM FOR SUSCEPTIBILITY TESTING OF COLISTIN BY THE DIFFUSION METHOD

[0002] Technical Field

[0003] The invention relates to a novel and specialised solid growth medium for use in susceptibility testing of polymyxins, such as colistin, using the diffusion method.

[0004] Prior Art

[0005] Infections caused by antibiotic-resistant bacteria are becoming increasingly common worldwide. In addition, multidrug-resistant, extensively drug-resistant and all drug-resistant infectious bacteria have been increasing in recent years. The number of antibiotics that can be used in the treatment of infections caused by such resistant bacteria is very limited. Among the antibiotics that can be used in infections caused by resistant gram-negative bacteria, one of the most important options is colistin (polymyxin E). In the last few years, bacteria resistant to colistin have also been reported. Therefore, colistin is not used alone in treatment and is usually combined with another antibiotic. However, antibiotic susceptibility testing for colistin remains a global problem. International guidelines such as EUCAST and CLSI currently state that the reference method for colistin susceptibility testing is the liquid microdilution method. Although automated antibiotic susceptibility testing systems are used, it is emphasised that they should be confirmed by liquid microdilution method and diffusion tests should not be used in colistin susceptibility testing.

[0006] There is not yet an economical, practical and scientifically valid susceptibility test method similar to diffusion tests (such as gradient strip test and disc diffusion test), which are widely used all over the world, that can be easily used in every laboratory. Because in the current practice, colistin does not diffuse sufficiently in agar-based media (such as Mueller Hinton agar) used for solidification of media due to its molecular structure. Therefore, diffusion tests have no scientific validity in colistin susceptibility tests.

[0007] The poor diffusion of colistin in agar results in high error rates compared to the liquid microdilution test for minimum inhibitory concentration determination. Other methods such as automated antibiotic susceptibility systems, gradient strip test and agar dilution are also unreliable in determining colistin susceptibility. Currently, there is no standardised disc diffusion test for colistin susceptibility testing in clinical microbiology laboratories. Currently, only one study in the literature (Uwizeyimana ID) modified Mueller-Hinton agar medium in an attempt to ensure colistin diffusion. In this study, protamine was used and the proportion of agar in the medium was reduced. The fact that protamine itself has a significant antibacterial effect and the technical application difficulties caused by the soft consistency of the medium due to the reduction of the agar ratio are very important disadvantages (Honda M, 2020, AzizM, 2019). These problems are major obstacles to the widespread use of protamine-modified Mueller-Hinton medium.

[0008] Detailed Description of the Invention

[0009] The invention relates to a solid growth medium in which colistin can diffuse. In the invention, since agar is not used solely to solidify the medium, the problem of non-diffusibility of colistin is solved. In the invention, a special colistin antibiotic susceptibility test medium has been developed in which colistin disc diffusion tests can be performed by using silica gel, carboxymethyl cellulose or gelatin in different ratios to the agar-based medium used to solidify the medium. Thus, colistin susceptibility can be tested in medical microbiology laboratories all over the world with an easy and scientifically validated method. The same is true for other polymyxins. Therefore, this medium has been developed for polymyxin B and other polymyxins in addition to colistin.

[0010] Figure 1. Colistin (Polymyxin E), Formula: C52H98N16O13, Molar mass: 1155.45 g / mol

[0011] Figure 2. Polymyxin B, Formula: CseHiooNieOr / S, Molar mass: 1301.57 g / mol In the invention, a simple disc diffusion test medium and method for susceptibility testing of colistin and other polymyxins has been developed by to improve the diffusion of colistin and other polymyxins in agar modifying the Mueller-Hinton medium used in antibiotic susceptibility testing by disc diffusion method. This medium allows the determination of antibiotic susceptibilities of many gram negative bacteria such as E.coli, Klebsiella pneumoniae, Pseudomona aeruginosa and Acinetobacter baumannii complex by disc diffusion method. However, there is a need for standardisation of the reference method, liquid microdilution tests, in colistin resistance mediated by the mcr gene. In order to determine the resistance mediated by the mcr resistance gene, phosphoethanolamine transferase inhibitors such as EDTA and Dipicolinic acid should be used. The medium of the invention is also suitable for the detection of colistin resistance mediated by the mcr resistance gene using the mentioned chemicals.

[0012] In the present invention; the medium does not have a tight molecular structure that does not allow some molecules to pass / diffuse like agar, but contains looser and larger pore structures. In addition to the large molecular structure, it is also suggested that colistin diffuses poorly into the agar due to electrostatic interactions with the acid or sulphate groups of the agar, resulting in smaller inhibition zone diameters and thus causing false colistin resistant / sensitive results. In the invention, colistin (polymyxin E) and other antibiotics with large molecular structure (other polymyxins) are enabled to disperse / diffuse in the solid medium. This is because agar is not used alone in the composition of the medium. Silica gel, carboxymethyl cellulose or gelatin were added to the agar-based medium used to solidify the medium at various ratios to obtain a looser and larger pore structure than agar. As a result, the problem of non-diffusibility of colistin was solved and a special solid medium suitable for a diffusion test method for colistin was developed.

[0013] Necessary comparisons and trials were performed and results were obtained in accordance with liquid microdilution, which is the reference method according to the international guidelines. In the modified Mueller-Hinton medium (Mueller-Hinton medium with silica gel and / or carboxymethyl cellulose and / or gelatin added) specified in the invention, inhibition zone diameters >11 mm were found to be susceptible in the liquid microdilution method.

[0014] Today, silica gel products of various brands (Merck, Sephadex and Zetadex etc.) and various sizes are used for different purposes. While the particle sizes of the silica gel beads in these products are 20-150 pm in dry state, the sizes of these particles are 15-610 pm when swollen in liquid medium. These silica gel beads are usually used by swelling in liquid medium. The degree of this swelling is also related to the liquid absorption volume of the beads. As a result, these silica gel particles become jelly-like or stiffer when swollen. Some voids are formed between these silica gel beads. In other uses of the present invention, the cavities, in other words, the pores and their sizes, which occur when the silica gel beads swell, are utilised. Silica gel combinations are selected according to the substances to be filtered (which are desired to pass through these pores or not).

[0015] Polymyxins (Polymyxin E / Colistin and Polymyxin B and others) have large structures and cannot pass through the pores and openings in conventional agar-based solid media. Therefore, the cheapest and most widely used antibiotic susceptibility test method cannot be used for these polymyxins. Instead, more expensive and more labour-intensive methods are used. In this invention, silica gel, carboxymethyl cellulose or gelatin is added at various ratios to the Mueller- Hinton agar medium used in antibiotic susceptibility tests by disc diffusion method in order to ensure that polymyxins (Polymyxin E / Colistin and Polymyxin B and others) disperse / diffuse in agar like other antibiotics. In one embodiment of the invention, silica gel beads are used to loosen the tight structure of the agar to allow diffusion of colistin and to enlarge small pore structures. In the invention, during the preparation of the medium, silica gel beads of different sizes and in different proportions are mixed / added to the powder medium at the stage before sterilisation by autoclave. The solid medium contains 1.5% to 2% agar by weight. Mueller-Hinton agar medium also contains the same percentage of agar. Silica gel beads with a dry particle size of 20-150 pm are mixed into this medium at a rate ranging from 0.001% to 1% by weight. The silica gel beads are kept for 1-3 hours depending on the ambient temperature for swelling. Then, standard autoclave sterilisation procedures are continued and the media are prepared.

[0016] In the present invention, carboxymethyl cellulose or gelatin can be used together with the silica gel beads, or these two substances can be added to the agar alone. Carboxymethyl cellulose absorbs the liquid in the medium and passes into hydrogel form. Hydrogel structures are three- dimensional polymeric network structures that can be prepared by physical, chemical or radiation cross-linking. These structures hold water tens of times more in weight than itself. They also have the property of insolubility. The pores in their structure allow the loading of compounds into the gel matrix and subsequent release of molecules of different sizes throughout the gel network at a rate dependent on the diffusion coefficient. Polysaccharide-based hydrogels can be widely used in various applications. Cellulose is an environmentally friendly polysaccharide, an alternative to traditional materials and has a wide range of applications. Today, hydrogels based on cellulose derivatives are used in the pharmaceutical industry and agriculture. Cellulose and its derivatives are used in the preparation of hydrogels because they have a large number of hydroxyl groups. Among these, cellulose ethers include two main cellulose derivatives. Cellulose ethers are generally hydrophilic and convert to hydrogels when exposed to water. In a cellulose-based hydrogel, the presence of sodium carboxymethyl cellulose provides swelling ability. In another embodiment of the invention, carboxymethyl cellulose is added to Mueller-Hinton agar medium at a ratio ranging from 0.1% to 1.5 % by weight. The medium is kept at room temperature for 10-90 minutes for hydrogel formation and then standard autoclave sterilisation procedures are continued and the media are prepared. The principle here is the same as in silica gel.

[0017] In this invention, gelatine was also used to loosen the stiffness of the agar. It has been known for many years that gelatine can be used to solidify media. However, since some bacteria have the ability to melt gelatin and gelatin has the ability to melt and solidify at low temperatures (18- 20°C), it is not widely used alone. In another embodiment of the invention, when preparing Mueller-Hinton agar medium, 0.1% to 4% by weight of type A or type B gelatin is added. The other steps are applied in the same manner.

[0018] In the invention, substances such as silica gel, carboxymethyl cellulose or gelatin are added to the Mueller-Hinton agar medium, which is widely used in antibiotic susceptibility testing in the world, according to the above-mentioned recipe and in the specified proportions. The mixed Mueller-Hinton agar medium is poured into petri dishes after being removed from the autoclave. After solidification, at least one petri dish is placed in an oven at 25-37°C and incubated for 16- 18 hours to check for contamination. Media that are found to be uncontaminated can be used for two weeks provided that they are stored at +4°C. The standard Kirby-Bauer disc diffusion method procedure is performed using the ready -to-use Mueller-Hinton agar medium in the form of a mixture. At the end of the incubation, the inhibition zone diameters (area without bacterial growth) formed around the antibiotic discs are measured. In the modified Mueller-Hinton medium (Mueller-Hinton medium with silica gel and / or carboxymethyl cellulose and / or gelatin added) specified in the invention, inhibition zone diameters >11mm were found to be sensitive in the liquid microdilution method.

[0019] International guidelines such as EUCAST and CLSI state that the reference method for colistin susceptibility testing is the liquid microdilution method. Although automated antibiotic susceptibility testing systems are used, it is emphasised that they should be confirmed by liquid microdilution method and diffusion tests should not be used in colistin susceptibility tests. Broth microdilution test can be performed inhouse by purchasing pure colistin and microplates together with other consumables. However, since it requires intensive labour and time, it is difficult to use in routine practice. There are several commercial ready-made microbroth dilution tests developed for colistin susceptibility testing available in the market. However, these are both very expensive and difficult to apply to individual samples. To summarise briefly, there are problems regarding susceptibility testing for colistin and other polymyxins. There is not yet an economical, practical and scientifically valid susceptibility test method that can be easily used in every laboratory similar to diffusion tests (such as gradient strip test and disc diffusion test) which are widely used all over the world.

[0020] Table 1. Sephadex silica gel specifications.

[0021] Table 2. Zetadex-25 ve Zetadex-50 silica gel specifications.

[0022] References:

[0023] 1. Uwizeyimana JD, Kim D, Lee H, Byun JH, Yong D. Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar. Ann Lab Med. 2020 Jul;40(4):306-311. doi: 10.3343 / alm.2020.40.4.306. PMID: 32067429; PMCID: PMC7054692.

[0024] 2. Honda M, Matsumoto M, Aizawa M. Potential Application of Protamine for Antimicrobial Biomaterials in Bone Tissue Engineering. Int J Mol Sci. 2020 Jun 19;21(12):4368. doi: 10.3390 / ijms21124368. PMID: 32575446; PMCID: PMC7352774.

[0025] 3. Aziz M, Garduno R, Mirani ZA, Baqai R, Sheikh AS, Nazir H, Raza Y, Ayaz M, Kazmi SU. Determination of antimicrobial effect of protamine by transmission electron microscopy and SDS PAGE on Pseudomonas aeruginosa isolates from diabetic foot infection. Iran J Basic Med Sci. 2019 Jul;22(7): 827-832. doi: 10.22038 / ijbms.2019.32414.7989. PMID: 32373306; PMCID: PMC7196347.

[0026] 4. https: / / echa.europa.eu / documents / 10162 / 5db3c0ed-7c4e-4379-8b9a-37aldf570519

Claims

CLAIMS1. Susceptibility testing growth medium by the diffusion method for polymyxins, characterised by the presence of agar and a thickening agent.

2. A growth medium according to claim 1 characterized in that the polymyxin is colistin.

3. A growth medium according to claim 1 characterized in that the thickening agent is silica gel, carboxymethyl cellulose, gelatine or a mixture thereof.

4. A growth medium according to claim 3 characterized by comprising 1.5% to 2% agar by weight and 0.001% to 1% silica gel by weight.

5. A growth medium according to claim 4 characterized in that the silica gel is in bead form having a particle size of 20-150 pm.

6. A growth medium according to claim 3 characterized by comprising 1.5% to 2% agar by weight and 0.1% to 1.5% carboxymethyl cellulose by weight.

7. A growth medium according to claim 3 characterized by comprising 1.5% to 2% agar by weight and 0.1% to 4% type A or type B gelatine by weight.

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