|Year : 2021 | Volume
| Issue : 1 | Page : 19-25
Validation of a novel medium for drug susceptibility testing of Mycobacterium Tuberculosis against First- and Second-Line drugs: AYC.2.2 Agar and AYC.2.1 Broth
Ahmet Yilmaz Coban
Akdeniz University Tuberculosis Research Center; Department of Nutrition and Dietetics, Akdeniz University Faculty of Health Sciences; Department of Medical Biotechnology, Akdeniz University Institute of Health Sciences, Antalya, Turkey
|Date of Submission||26-Oct-2020|
|Date of Acceptance||30-Nov-2020|
|Date of Web Publication||28-Feb-2021|
Ahmet Yilmaz Coban
Akdeniz University Tuberculosis Research Center; Department of Nutrition and Dietetics, Akdeniz University Faculty of Health Sciences, Department of Medical Biotechnology, Akdeniz University, Institute of Health Sciences, Antalya
Source of Support: None, Conflict of Interest: None
Background: The aim of this study was the validation of AYC.2.2 agar and AYC.2.1 broth for the breakpoint values of first- and second-line drugs for Mycobacterium tuberculosis. Method: A total of 12 isolates including 5 reference strains and 7 well-defined clinical isolates were tested for their antituberculosis susceptibilities. Inhibitory effects of first- and second-line antituberculous drugs including isoniazid, rifampicin, streptomycin, ethambutol, amikacin, capreomycin, kanamycin, para-aminosalicylic acid, ethionamide, rifabutin, ofloxacin, levofloxacin, and moxifloxacin were tested. Results: According to the minimal inhibitory concentration values obtained in 7H10 agar, 7H9-S broth, AYC.2.2 agar, and AYC.2.1 broth, category agreement is 100%, and very major discrepancy (MAD), MAD, and minor discrepancy ratios were determined as 0 for all drugs. Conclusion: It was concluded that breakpoint values by CLSI recommendation for 7H10 agar can be also used for AYC.2.2 agar and AYC.2.1 broth. In addition, further multicenter studies are needed to use the new medium in routine mycobacteriology laboratories.
Keywords: AYC.2.1 broth, AYC.2.2 agar, breakpoint value, first- and second-line drugs, Mycobacterium tuberculosis, susceptibility testing
|How to cite this article:|
Coban AY. Validation of a novel medium for drug susceptibility testing of Mycobacterium Tuberculosis against First- and Second-Line drugs: AYC.2.2 Agar and AYC.2.1 Broth. Int J Mycobacteriol 2021;10:19-25
|How to cite this URL:|
Coban AY. Validation of a novel medium for drug susceptibility testing of Mycobacterium Tuberculosis against First- and Second-Line drugs: AYC.2.2 Agar and AYC.2.1 Broth. Int J Mycobacteriol [serial online] 2021 [cited 2021 Jun 14];10:19-25. Available from: https://www.ijmyco.org/text.asp?2021/10/1/19/310500
| Introduction|| |
Tuberculosis (TB) is one of the oldest health problems that still cannot be resolved worldwide and is the 10th important cause of death in the world annually. Approximately 10 million people were estimated to fell ill TB in 2018 across the globe. Besides, 1.2 million human immunodeficiency virus (HIV)-negative people and 251,000 HIV-positive people are estimated to have died from TB in 2018.
The most important and time-consuming aspect of combating TB infection is the early diagnosis and treatment of patients. Early determination of antituberculosis resistance is quite important for the appropriate treatment of cases. Today, various methods are using in the determination of drug resistance isolates. Middlebrook 7H10-11 agar and LJ media are recommended by CLSI for susceptibility testing. Middlebrook 7H10-11 agar is expensive, due to additional enrichment with oleic acid, albumin, dextrose, and catalase (OADC) which should be used within 7H10 and 11 agars. Despite the low cost, preparation of LJ media with antibiotics is quite challenging and the results can be obtained in a long time (4–6 weeks). There are also commercial systems available that give faster results, such as Bactec MGIT 960 and TK-Medium; however, due to the necessity for new equipment installation and high costs of these systems, practically they cannot be used in laboratories with limited sources.,,
Even though the existing methods mentioned above are applied, it is aimed to develop a new medium that may be an alternative to them. While developing the medium, enrichers such as peptone and some chemicals and sheep serum for protein content, which are needed for the growth of microorganisms, were also used. The media obtained has been developed as a medium that will provide an advantage in terms of cost and can be an alternative to existing media.
The AYC.2.2 agar and AYC.2.1 broth (PCT/TR2018/050884), which have been newly developed and applied for the Patent Cooperation Treaty, are low cost promising media for resistance determination of TB. The present study aims to test AYC.2.2 agar and AYC.2.1 broth for primary and secondary antituberculosis drugs and to define breakpoint concentrations of 13 antituberculosis drugs for both media.
| Method|| |
All preparation and experiments of this study had been done in a level 3 biosafety laboratory. A total of 12 Mycobacterium tuberculosis strains including M. tuberculosis H37Rv, ATCC35822 (INH resistant), ATCC35838 (RIF resistant), ATCC35820 (STM resistant), and ATCC35837 (EMB resistant) were tested. The remaining 7 isolates were obtained from the T. C. Ministry of Health Public Health TB Reference Laboratory. Of these well-defined isolates, 4 were susceptible to primary antituberculosis drugs and 3 were resistant. One of the 3 resistant isolates was isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), ethambutol (EMB), and ofloxacin (OFX) resistant: one was resistant to INH, RIF, PZA, EMB, OFL and STM and the other was resistant to AMK, KAN, and CAP. Fresh cultures of isolates were prepared on the LJ medium before they were tested.
Preparation of media
AYC.2.2 agar and AYC.2.1 broth include L-asparagine, monopotassium phosphate, magnesium sulfate, magnesium citrate, peptone, and glycerol. AYC.2.2 agar and AYC.2.1 broth were prepared according to the producer's recommendations, and 5% inactivated sheep serum (Sigma-Aldrich) was added to medium when cooled to 50°C. The 7H10 agar and 7H9-S broth were prepared according to the manufacturer's recommendations, and when cooled to 50°C, OADC was added as 10%. In addition, 0.1% casitone was added into 7H9-S broth.
Preparation of antibiotics
The antibiotics tested are INH, RIF, EMB, STM, AMK, CAP, ETH, KAN, LEV, MOX, OFX, PAS, and RIFA. Stocks at a concentration of 10,000 μg/ml were prepared for each antibiotic. The prepared stock solutions were sterilized by filtration and stored at −80°C in small quantities until use.
Preparation of Middlebrook 7H10 agar as a reference method and AYC.2.2 agar for minimal inhibitory concentration determination
Each antibiotic was tested at 7 different concentrations. Tubes containing the concentrations INH: 0.03–2 μg/ml, RIF: 0.06–4 μg/ml, EMB: 0.5–32 μg/ml, STM: 0.25–16 μg/ml, AMK: 0.5–32 μg/ml, CAP: 0.6–40 μg/ml, ETH: 0.3–20 μg/ml, KAN: 0.3–20 μg/ml, LEV: 0.06–4 μg/ml, MOX: 0.06–4 μg/ml, OFX: 0.125–8 μg/ml, PAS: 0.125–8 μg/ml, and RIFA: 0.06–4 μg/ml were prepared for each antibiotic. One antibiotic-free growth control medium tube was prepared for each bacterium. The medium was aliquoted in 5 ml sterile screw-cap tubes and solidified in slant position, and they were stored at 4°C until use. The storage period did not exceed 4 weeks.
Preparation of resazurin-based microplate method plates in AYC.2.1 broth and 7H9-S broth for minimal inhibitory concentration determination
Minimal inhibitory concentration (MIC) determination in 7H9-S broth (0.1% casitone and 10% OADC) and AYC.2.1 broth was performed by the resazurin-based microplate method (REMA). Antibiotic concentrations prepared for this purpose were the same as those stated in the reference method above. The plates were prepared in 100 μl AYC.2.1 broth and 7H9-S broth in 96-well plates with two-fold dilutions. A growth control well without antibiotics was also prepared for each strain tested. The prepared plates were stored at −80°C until use.
Preparation of bacterial inoculum
In the current study, fresh cultures of bacteria were used from the LJ medium. Bacteria colonies were taken and transferred to tubes containing 5 ml saline and sterile glass beads. Tubes were vortexed for 1 min. The aerosol and large particles were left in an upright position at room temperature for 1 h to settle. Afterward, the supernatant was taken and transferred to another tube and adjusted to McFarland no. 1 standard.
Application of tests on solid media for minimal inhibitory concentration determination
Susceptibility testing was performed as the reference method. Shortly, after McFarland no. 1 standard bacteria 1:100 diluted, 100 μl of diluted inoculum was inoculated in test tubes with antibiotics and growth control tube without antibiotics. Tubes containing 7H10 agar were incubated at 37°C and 5%–10% CO2, while tubes containing AYC.2.2 agar were incubated at 37°C. Tubes were examined for growth every 3 days for the 1st week and then once a week. At the end of the 21-day incubation period, tests were concluded for antibiotics with sufficient growth in the growth control tube. The concentration in the last tube where growth was not observed was reported as the MIC value.
Application of resazurin-based microplate method in AYC.2.1 broth and 7H9-S broth for minimal inhibitory concentration determination
The prepared bacterial inoculum was diluted 1:10 with 7H9-S broth for the test to be performed on the 7H9-S broth and then inoculated as 100 μl in each well. Plates were incubated at 37°C. Similarly, to perform the AYC.2.1 broth test, the bacterial inoculum was diluted with AYC.2.1 broth in 1:10 and then inoculated in 100 μl per wells. Plates were incubated at 37°C. On the 7th day of incubation, 30 μl of 0.02% resazurin solution was added in all wells of the test plates, and the plates were re-incubated at 37°C. The tests were concluded when the blue color turned red in the growth control well at the end of the incubation (1 or 2 days). The color turning from blue to red is an indicator of growth in the well. Therefore, the last well that the color did not turn red was reported as the MIC value.
Analysis of results
The results were evaluated according to the Food and Drug Administration (FDA) guidance. The results obtained in 7H10 agar, which is the reference method, were compared in terms of MICs obtained with AYC.2.2 agar, AYC.2.1 broth, and 7H9-S broth, and essential agreement (EA), category agreement (CA), major discrepancy (MAD), minor discrepancy (MID), and very MAD (VMD) ratios were calculated for each antibiotic.
| Results|| |
In this study, 5 reference isolates and 7 well-defined clinical isolates were tested. All tests were performed two times, and the same MIC values were detected. The MIC values obtained are summarized in [Table 1],[Table 2],[Table 3],[Table 4]. In the study, the MIC values obtained with AYC.2.2 agar, AYC.2.1 broth, and 7H9 broth were compared with those obtained with 7H10 agar [Table 5]. The MIC test performed on 7H10 agar was the reference method (gold standard test). The MIC results were evaluated according to the formulas defined by the FDA. Accordingly, if MIC values are within ± 1 dilution, it is defined as EA and is calculated by dividing the total number of concordant isolates by the total number of isolates tested. A new method is categorically defined as concordant if it produces identical results from an isolate, which were defined as resistant or sensitive with the reference method according to the MIC values obtained. CA is calculated by dividing the number of categorically concordance isolates by the total number of bacteria tested. Furthermore, VMD, MAD, and MID ratios were calculated as defined by the FDA.
|Table 1: Minimal inhibitory concentration values obtained in 7H10 agar, AYC.2.2 agar, AYC.2.1 broth, and 7H9-S broth|
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|Table 2: Minimal inhibitory concentration values obtained in 7H10 agar, AYC.2.2 agar, AYC.2.1 broth, and 7H9-S broth|
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|Table 3: Minimal inhibitory concentration values obtained in 7H10 agar, AYC.2.2 agar, AYC.2.1 broth, and 7H9-S broth|
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|Table 4: Minimal inhibitory concentration values obtained in 7H10 agar, AYC.2.2 agar, AYC.2.1 broth, and 7H9-S broth|
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|Table 5: Comparing minimal inhibitory concentration values obtained in 7H10 agar with minimal inhibitory concentrations obtained from AYC.2.2, AYC.2.1, and 7H9 broth according to FDA criteria|
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When the MIC values were obtained with the reference method (7H10 agar) and AYC.2.2 agar, AYC.2.1 broth, and 7H9 broth compared, the CA was 100% for all antibiotics [Table 5]. Meaning that the results found to be susceptible or resistant with 7H10 agar were found to be the same with AYC.2.2. EA was 91.66%–100% for all antibiotics except RIF. EA was 66.66% for RIF with AYC.2.1 broth [Table 5].
VMD, MAD, and MID ratios were detected as 0% when the MIC values were obtained by the reference method, and three media were compared [Table 5]. After the validation study, it was found appropriate to use the breakpoint values recommended by CLSI in 7H10 agar for both AYC.2.2 agar and AYC.2.1 broth.
| Discussion|| |
LJ medium is frequently used for the growth of mycobacteria from clinical specimens, while 7H9 broth is used in the Bactec MGIT 960 system, which is the most widely used automated system; drug susceptibility tests of M. tuberculosis isolates are performed on LJ media, Middlebrook 7H10, and 11 agar media. Newly developed rapid susceptibility tests including reassuring microplate method, nitrate reductase assay, and crystal violet decolorization test are performed in Middlebrook 7H9 broth.,,
In recent years, several new media have been studied for isolation of mycobacteria (especially isolation of M. tuberculosis from clinical samples) and antibiotic susceptibilities. In this context, the most limiting factor is cost and time features. Commercially available systems on the market can determine the susceptibility test in less time but at a higher cost, however, traditional methods consume much more time, but they are inexpensive in return. In 2003, it was shown that blood agar can be used to isolate M. tuberculosis from clinical samples. In a subsequent study, blood agar is cost-effective in the growth of mycobacteria from clinical specimens. It was also shown that M. tuberculosis grows on blood agar earlier than other solid media in terms of duration.,,,,
After demonstrating M. tuberculosis isolates that may grow on blood agar, studies have also been conducted for antibiotic susceptibility tests. In these studies, it was stated that blood agar could be used for both primary and secondary antituberculosis drug susceptibility testing. It is even reported that blood agar can be used for direct susceptibility testing from clinical samples.,,,,, The blood agar was also validated in 2013 by Coban for primary antituberculosis drugs.
The blood agar medium is a red-color medium and is not transparent like 7H10 and 11 agar. Therefore, it may be challenging to observe colonies. Coban et al. suggested using sheep serum instead of sheep blood in the blood agar base medium to eliminate this disadvantage.
There are also newly developed media in this process. Recently, it was developed a novel media as chlorhexidine agar medium (MOD9) and it was reported that M. tuberculosis was isolated from clinical samples in a shorter time on MOD9 medium., There are also several similarly developed media for this purpose.,,
AYC.2.1 broth and AYC.2.2 agar are also newly developed media. The medium contains essential substances for the growth of mycobacteria. Furthermore, there is sheep serum as an enriching supplement. In this study, MIC results obtained in 7H10 agar were compared with MICs obtained from AYC.2.2 agar, AYC.2.1 broth (with REMA method), and 7H9 broth (with REMA method). In the present study, both primary and secondary antituberculosis 13 antibiotics were tested and breakpoint values of these antibiotics were defined. Complete concordance with all media had been detected in all antibiotics. It was concluded that breakpoint values by CLSI recommendation for 7H10 agar can be also used for AYC.2.2 agar and AYC.2.1 broth.
Newly developed media contains peptone as basic ingredient and L-asparagine, monopotassium phosphate, magnesium sulfate, magnesium citrate, and glycerol chemicals. Sheep serum was added as an enrichment. It contains approximately 7.5% total protein (albumin, alpha globulin, beta globulin, and gamma globulin), cholesterol, iron, glucose, triglyceride, etc., in sheep serum. These substances are used by microorganisms to speed up and facilitate their growth. OADC supplements are also used as enrichers for Middlebrook media, and it contains OADC.
The OADC supplement should be added to a final concentration of 10% in 7H10 and 11 agars. The OADC is commercialized in its liquid form and the shelf life of this product is relatively short, 365 days after production. Preparation of AYC.2.1 and AYC.2.2 medium requires enrichment with sheep serum which facilitates bacterial growth. Sheep serum should be added to a final concentration of 5%. It is well known that sheep serum can be stored at −20°C without losing its performance characteristics up to 5 years. In addition, OADC is added to the medium at a rate of 10% whereas sheep serum at a rate of 5%. As a result, sheep serum is used twice less in quantity.
| Conclusion|| |
After this validation study, it was found appropriate to use the breakpoint values recommended by CLSI in 7H10 agar for both AYC.2.2 agar and AYC.2.1 broth. These potential media, AYC.2.2 agar, and AYC.2.1 broth should be tested with more clinical isolates at the breakpoint values specified in the following process. Besides, the developed medium may inevitably be an alternative to the existing media, though multicenter evaluations and further studies are needed.
The author appreciates M. Pooya Salehi Moharer for providing support to this article.
Financial support and sponsorship
This study was funded by the Scientific and Technological Research Council of Turkey (TUBITAK) (No: 114S798).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]