STANDARDIZATION STUDY FOR AGAR WELL DIFFUSION METHOD

Öz

Antibiotic sensitivity tests are important to determine antibiotics that can be used against disease-causing microorganisms. Although different standardization studies for disk diffusion and MIC tests were established by several organizations, no standardization study was done for agar well diffusion method. Standardization for disk diffusion method, antibiogram, was done first by World Health Organization (WHO). Today many organizations including, The European Committee on Antimicrobial Susceptibility Testing (EUCAST) in Europe, Clinical and Laboratory Standards Institute (CLSI) in USA, and Standardization of Antibiotic Sensitivity Tests (ADTS) group of Turkish Microbiology Society (TMC) in Turkey developed their own standardization methods. TMC advises the use of EUCAST protocols for studies in Turkey.

The aim of the present study was standardization of agar well diffusion method. Disk diffusion method from EUCAST was used as a reference method to compare results of agar well diffusion test. For both disk diffusion method as well as agar well diffusion test 0.5 McFarland bacterial suspension and Mueller Hinton Agar (MHA) media were used. For agar well diffusion test 3 different thicknesses, 4 mm, 6 mm and 8 mm, of MH agar was used. Also three different well diameter, 4 mm, 6 mm and 8 mm were tested. All tests were performed as three repetitions. Two antibiotics were used for disk diffusion tests and agar well diffusion tests. The concentration of erythromycin and chloramphenicol were 15 µg and 10 µg, respectively. A Gram positive and a Gram negative strain were used for susceptibility tests, S. aureus ATCC 25923 and E. coli DH10B strains, respectively. All of plates were incubated at 37°C for 16-18 hours. At the end of incubation, the inhibition zones around wells were measured.

The inhibition zones of S. aureus ATCC 25923 with erythromycin disk were 29, 30 and 31 mm on 4, 6 and 8 mm agar, respectively. Agar well diffusion with 4, 6 and 8 mm MHA media, erythromycin inhibition zones were 21, 25 and 29 mm, 22, 26 and 30 mm, and 23, 27 and 31 mm, respectively. The inhibition zones of E. coli DH10B with chloramphenicol disk were 25, 26 and 27 mm on 4, 6 and 8 mm agar, respectively. Agar well diffusion with 4, 6 and 8 mm MHA media, chloramphenicol inhibition zones were 5, 6 and 7 mm, 15, 16 and 17 mm, and 25, 26 and 27 mm, respectively. It was observed that the disk diffusion inhibition zones were in accordance with the results of 8 mm agar well zone, in every MH agar thicknesses. As the standard for disk diffusion test the MHA thickness is 4 mm, the agar well diameter for this thickness is 8 mm. So for standardization of agar well diffusion tests, the MHA thickness should be 4 mm and the diameter should be 8 mm.  

A standardization method for agar well diffusion test is necessary to compare results obtained elsewhere. Our study is the first study for standardization of agar well diffusion test. Standardization assays should be done by other laboratories to determine a worldwide usable and comparable agar well diffusion method. 

Anahtar Kelimeler

• Agarwell,Standardization,Media thickness,Well diameter

AGAR WELL DİFÜZYON YÖNTEMİNDE STANDARDİZASYON ÇALIŞMASI

Öz

Antibiyotik duyarlılık testleri hastalık etkeni mikroorganizmalara karşı kullanılabilecek antibiyotiklerin belirlenmesinde önemlidir. Antibiyogram testlerinde farklı kuruluşlar tarafından standardizasyon çalışması yapılmasına karşın agar well testleri için standardizasyon çalışmasının yapılmadığı görülmüş ve bu çalışmada disk difüzyon testiyle karşılaştırmalı standardizasyon çalışması yapılması amaçlanmıştır. Testler 0,5 McFarland bulanıklıkta bakteri süspansiyonu ile 3 farklı kalınlıkta (4, 6, 8 mm) Mueller Hinton agar besiyeri ve 3 farklı çapta kuyucuk (4, 6, 8 mm) kullanılarak üç tekrarlı olacak şekilde yürütülmüştür. S. aureus ATCC 25923 ve E. coli DH10B bakterileri için sırasıyla eritromisin (15µg) ve kloramfenikol (10µg) antibiyotikleri kullanılmıştır. Tüm petriler 16-18 saat, 37°C’de inkübe edilmiştir. İnkübasyon sonunda disklerin ve kuyucukların etraflarında oluşan inhibisyon zonları ölçülmüştür. Antibiyotik duyarlılık testinde kullanılan agar well yöntemi için yaptığımız standardizasyon çalışmasında en uygun Mueller Hinton Agar besiyeri kalınlığı ve kuyucuk çapı saptanmıştır. Agar kalınlığı ne olursa olsun, disk difüzyon zon çapıyla agar well kuyucuk zon çapının 8 mm kuyucukla eşleştiği görülmüştür. Agar kalınlığı arttıkça inhibisyon zonu artmaktadır. Disk difüzyon testinde standart 4 mm kalınlığındaki besiyeri olduğu için bu kalınlıktaki besiyerinde disk difüzyon sonucuna uyan inhibisyon zonu oluşturan kuyucuk çapı 8 mm olarak saptanmıştır. Standart ölçüm için 4 mm agar kalınlığı ve 8 mm kuyucuk çapı olmalıdır.

Çalışmamız agar well difüzyon konusundaki ilk standardizasyon çalışmasıdır. Diğer laboratuvarların benzer standardizasyon çalışmaları yapmaları halinde agar well difüzyon yöntemini tüm dünyada geçerli olabilecek bir standarda kavuşturabiliriz.

Anahtar Kelimeler

• Agarwell,Standardizasyon,Besiyeri kalınlığı,Kuyucuk çapı

Kaynakça

1. 1. Athanassiadis B., Abbott P. V., George N., & Walsh L. J., (2009). An in vitro study of the antimicrobial activity of some endodontic medicaments and their bases using an agar well diffusion assay. Australian dental journal, 54(2), 141-146.
2. 2. Aadesariya M. K., Gauni B. M., Duggirala S. M., Ram V. R., & Vyas S. J. (2017). Antibacterial activity of different solvent leaves extracts of abutilon pannosum and grewia tenax against different type of gram positive and gram negatıve bacteria by agar well diffusion method. World Journal of Pharmaceutical Research, Volume 6, Issue 16, 1259-1274.
3. 3. Boorn K. L., Khor Y. Y., Sweetman E., Tan F., Heard T. A., & Hammer K. A. (2010). Antimicrobial activity of honey from the stingless bee Trigona carbon aria determined by agar diffusion, agardilution, broth microdilution and time‐kill methodology. Journal of applied microbiology, 108(5), 1534-1543.
4. 4. Boyanova L., Gergova G., Nikolov R., Derejian S., Lazarova E., Katsarov N., & Krastev Z., (2005). Activity of Bulgarian propolis against 94 Helicobacter pylori strains in vitro by agar-well diffusion, agardiluti on and disc diffusion methods. Journal of medical microbiology, 54(5), 481-483.
5. 5. Balouiri M., Sadiki M., & Ibnsouda S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of pharmaceutical analysis, 6(2), 71-79.
6. 6. Bauer A. W., Kirby W. M. M., Sherris J. C., & Turck M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology, 45(4_ts), 493-496.
7. 7. Courvalin P. (2006) Antibiyogram. Antibiogramme 2. Baskı. 8. Charannya S., Duraivel D., Padminee K., Poorni S., Nishanthine C., & Srinivasan M. R. (2018). Comparative evaluation of antimicrobial efficacy of silver nanoparticles and 2% chlorhexidine gluconate when used alone and in combination assessed using agar diffusion method: An In vitro study. Contemporary clinical dentistry, 9(Suppl 2), S 204.
8. 9. Devillers J., Steiman R. & Seigle-Murandi F. (1989). The usefulness of the agar-well diffusion method for assessing chemical toxicity to bacteria and fungi. Chemosphere, 19(10-11), 1693-1700.

9. 10. EUCAST (2019) Disk Diffusion Method for Antimicrobial Susceptibility Testing. Version 7.0 (January 2019).
10. 11. Gür D. (2016), Antibiyotik Duyarlılık Testleri, EUCAST: Uygulama, Yorum ve Uzman Kurallar. Türk Mikrobiyoloji Cemiyeti Dergisi, Cilt/Volume 46.
11. 12. Holder I. A. & Boyce S. T. (1994). Agar well diffusion assay testing of bacterial susceptibility to various antimicrobials in concentrations non-toxic for human cells in culture. Burns, 20(5), 426-429.
12. 13. Milletli-Sezgin F., Sevim E., & Sevim A. (2019). Enterokok Suşlarında Antibiyotik Duyarlılığı: CLSI ve EUCAST Disk Difüzyon Klinik Sınır Değer Yorumlarının Karşılaştırılması. Klimik Dergisi, 32(1), 35-9.
13. 14. Magaldi S., Mata-Essayag S., De Capriles C. H., Perez C., Colella M. T., Olaizola C. & Ontiveros Y. (2004). Well diffusion for antifungal susceptibility testing. International journal of infectious diseases, 8(1), 39-45.
14. 15. Rogers A. M. & Montville T. J. (1991). Improved agar diffusion assay for nisin quantification. Food Biotechnology, 5(2), 161-168.
15. 16. Ruiz M. V., Silva P. G. & Laciar A. L. (2009). Comparison of microplate, agar drop and well diffusion plate methods for evaluating hemolytic activity of Listeria monocytogenes. African Journal of Microbiology Research, 3(6), 319-324.
16. 17. Sümerkan B. (1996). Antibiyotik Duyarlılık Testleri ve Standardizasyon. Flora İnfeksiyon Hastalıkları ve Klinik Mikrobiyoloji Dergisi, 1(1), 24-30.
17. 18. World Health Organization. Expert Committee on Antibiotics & World Health Organization. ‎(1961). Standardization of methods for conducting microbic sensitivity tests: second report of the Expert Committee on Antibiotics [‎meeting held in Geneva from 11 to 16 July 1960]‎. World Health Organization.
18. 19. Vu H., McCoy L. F, Carino E., Washington J., Dang T., Villareal C., Rosenblatt J., Maness C., Goodheart R. & Heggers J. P. (2002). Burn wound infection susceptibilities to topical agents: The Nathan's agar well diffusion technique. P AND T, 27(8), 390-397.