Background and Objectives: Antibacterial susceptibility testing of clinical bacterial isolates through disk diffusion method plays a major role in antibacterial treatment. One of the main factors affecting the result of these tests is the type, structure and quality of the disks. The main objective of this study was to compare the agreement of antibiotic disks originated from three companies on Quicolor and Mueller-Hinton agar.



Materials and Methods: Quicolor and Mueller-Hinton agar media were used in disk diffusion method. Seventy clinical isolates from Enterobacteriaceae family (21 Klebsiella spp., 36 Escherichia coli, 1 Enterobacter spp. and 12 Shigella spp.) were investigated in the study. After obtaining data, the results were interpreted as resistant, sensitive or intermediate. Kappa coefficient measured the agreement of two media. Coefficient of variation (CV) was also calculated for antibiotic disks.



Results: The kappa agreement values for three types of antibiotic disks on Quicolor and Mueller-Hinton agar plates were good or excellent for all the examined antibiotics. CV values were also very satisfactory in the majority of cases.



Conclusion: Antibiotic disks from three manufacturers can successfully be used on both Quicolor and Mueller-Hinton agar plates

Abdi-Ali, Ahya

Rezaei Hemami, Mohsen

Saffari, Neda

Salmanzadeh-Ahrabi, Siavosh

English

PubMed

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307*Corresponding author: Siavosh Salmanadeh-Ahrabi, PhD, Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.Tel: +989336159153Fax: +98-21-88058912Email: salmanzadeh1@yahoo.comA comparison of antibiotic disks from different sources on Quicolor and Mueller-Hinton agar media in evaluation of antibacterial  susceptibility testingNeda Saffari1, Siavosh Salmanzadeh-Ahrabi2*, Ahya Abdi-Ali2, Mohsen Rezaei-Hemami3 1Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran2Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran3Institute of Health & Wellbeing, Health Economics & Health Technology Assessment, University of Glasgow, Glasgow, ScotlandReceived: February 2016, Accepted: June 2016ABSTRACT  Background and Objectives: Antibacterial susceptibility testing of clinical bacterial isolates through disk diffusion method plays a major role in antibacterial treatment. One of the main factors affecting the result of these tests is the type, structure and quality of the disks. The main objective of this study was to compare the agreement of antibiotic disks originated from three companies on Quicolor and Mueller-Hinton agar.Materials and Methods: Quicolor and Mueller-Hinton agar media were used in disk diffusion method. Seventy clinical isolates from Enterobacteriaceae family (21 Klebsiella spp., 36 Escherichia coli, 1 Enterobacter spp. and 12 Shigella spp.) were investigated in the study. After obtaining data, the results were interpreted as resistant, sensitive or intermediate. Kappa coefficient measured the agreement of two media. Coefficient of variation (CV) was also calculated for antibiotic disks.Results: The kappa agreement values for three types of antibiotic disks on Quicolor and Mueller-Hinton agar plates were good or excellent for all the examined antibiotics. CV values were also very satisfactory in the majority of cases.Conclusion: Antibiotic disks from three manufacturers can successfully be used on both Quicolor and Mueller-Hinton agar plates.Keywords: Antibiotic disks, Disk diffusion, Quicolor mediumVolume 8 Number 5 (October 2016) 307-311ORIGINAL ARTICLEINTRODUCTIONDiscovery of antibiotics was a tremendous help in quick and accurate treatment of infectious diseases (1). Disk diffusion method is widely used to detect the susceptibility of bacterial isolates to antibiotics. Colorimetric media have also been developed for rapid antibacterial susceptibility testing of bacteria instead of Mueller-Hinton agar plates. Quicolor (QC) (Salubris Inc., Massachusetts, USA) is a medium for colorimetric and rapid antibacterial susceptibility testing. It is based on a rapid culture medium that indicates early growth of bacterial through changes in the color. Since the results are available within 3.5-6 hours after inoculation, it may have a signif-icant impact on reduction of hospitalization time, http://ijm.tums.ac.irNeDA SAffARI eT AL .                                                                                                            308           IRAN. J. MICROBIOL.  Volume 8 Number 5 (October 2016) 307-311 total medical costs and even rate of mortality. Quicol-or is cost-effective, easy to use and do not require any special instruments. Use of antibiotic disks is nec-essary for both mentioned media. According to the Clinical and Laboratory Standards institute (CLSI) guidelines, interpretation of the results in standard disk diffusion method is on the basis of growth inhibition zone whereas for the Quicolor is based on the color changes according to the manufacturer’s instructions. The Kirby Bauer technique (2) for disk susceptibility testing has been recommended by the CLSI (3) which has been approved by Food and Drug Administration (FDA) and is also recommended by WHO (4, 5). Thus, the antibiotic sensitivity test re-port can have a strong effect on antibiotic consump-tion and hence on the factors that facilitate the emer-gence of antimicrobial drug resistance. Therefore, the test should be highly standardized using standard reagents, disks and appropriate strains as the quali-ty controls. The antibiotic disks themselves serve as key parameters in obtaining accurate and reproduc-ible results (6-8).Quality of disks and potency of their antibiotics, produced by different manufactures, must be ap-proved through three FDA,WHO and the US De-partment of National Health and Welfare (DNHW) (9). Different levels of antibiotic saturation may be chosen by the manufactures since some disks may be impregnated with more than 100% of the stated con-tent to compensate for loss of activity in the handling of disks (10).There are three main international standards for potency of antibiotic in the disks and all the manu-factures do not produce according to the same stan-dards. Their specifications have been summarized as follows: FDA specification 67-150%, WHO specifi-cation 75-135% and DNHW 90-125% of the stated concentration (11, 12).In this study, we investigated the quality and type of antibiotic disks from different origins and their effects on the results of antibacterial susceptibility testing. MATeRIALS AND MeTHODS    Bacterial isolates. Seventy clinical isolates from Enterobacteriaceae family (21 Klebsiella spp., 36 Escherichia coli, 1 Enterobacter spp and 12 Shigel-la spp.) were obtained from Pasteur Institute of Iran isolated from blood or urine samples during the years 2009-2010. Strains were confirmed through bio-chemical tests and stored at -70 °C for future use. E. coli ATCC 25922 strain was used as a quality control strain. Agar media. Dehydrated Quicolor ES agar medi-um (specific for Enterobacteriaceae and Staphylo-cocci) containing carbohydrates, peptones, dye indi-cator, vitamins, salts as well as Quicolor enrichment supplement were used. Mueller-Hinton agar plates were also prepared according to the manufacturer’s instructions.Antibiotics. Antibacterial susceptibility testing was done by standard disk diffusion method on Mueller-Hinton agar and Quicolor according to CLSI or manufacturers guidelines using disks from three different manufacturers; Rosco, Mast and Padtan Teb companies. The results were interpreted as re-sistant, sensitive or intermediate. Five types of an-tibiotics were selected including ciprofloxacin (CIP, 5µg), cefotaxime (CTX, 30µg), cefazolin (CZ, 30µg), meropenem (MEM, 10µg) and cotrimoxazole (SXT, 25µg). Three batches were used for each of five types in one experiment, one for antibiotics from mast, an-tibiotic from Rosco company (NEO-Sensitabs-Ros-co), and Padtan Teb.Disk diffusion method. Antibiotic susceptibility test was done according to Kirby Bauer method (2) using 2 mentioned media. All procedures and con-ditions were the same to ensure even in the cases of different zone diameters there is no error. The influ-encing factors such as type, depth and pH of the me-dium, type of the clinical strains, using the same bac-terial colony in order to make bacterial suspension, potency of antibiotic disks, accuracy of the cultiva-tion procedure were considered in the experiments (13, 14). Zone of inhibition in Quicolor plates was measured after 4-6 hours incubation whereas inhibi-tion zones in  MHA plates was measured after 18 to 24 hours (7). Statistical analysis. Statistical analysis was done by SPSS (v. 22) and Stata softwares. CV was calcu-lated for each antibiotic disk on both Quicolor and Mueller-Hinton agar plates. Data have been reported after four repeats of disk diffusion test. Reproduc-ibility of the results was considered unsatisfactory http://ijm.tums.ac.irCOMpARISON Of DffeReNT ANTIBIOTIC DISKSIRAN. J. MICROBIOL.  Volume 8 Number 5 (October 2016) 307-311               309 http://ijm.tums.ac.irif CV percentage of a disk was more than 5%. Sub-sequently Kappa values were calculated to inves-tigate the agreement of the results on both media (Table 3).Kappa values interpreted  in four groups: weak agreement for values among 0 to 0.25, moderate agreement for 0.25 to 0.5, good agreement for 0.5 to 0.75 and excellent agreement for 0.75 up to 1. In the other way, we stated percentage of agreement with turning Kappa values to percentage from 0 to 100. Kappa +1 indicate that two cases that compare with another act similar to another com-pletely. ReSULTSDisk diffusion test was done for all strains sepa-rately. Every time for one test quality control experi-ment was done for validation of other tests, antibiotic disks, cultures and overall. The results were accept-able and diameter of inhibition zone was in its range (Table 1 and 2).In this study, antibiotics originated from three manufactures, NEO-SENSITABS-Rosco (Den-mark), Mast (UK) and Padtan Teb (Iran) were com-pared. Kappa values have been presented in Table 3 indicating all excellent or good agreements ex-cept for meropenem on MHA plates that showed moderate agreement. In the other way, we turn Kappa values to agreement percentage (Tables 4 and 5).Four Mast disks, 3 Rosco tablets and 2 disks of Padtan Teb showed slightly higher CV values (great-er than 5%) when Mueller-Hinton agar medium was used. In the case of Quicolor plates, one Mast disk, 2 Rosco-tablets and 6 disks from Padtan Teb company showed slightly higher CV values (higher than 5%).Table 1. Quality control of antibiotic disks using Escherich-ia coli ATCC 25922 control strainAntibioticsCiprofloxacinCefotaximeCefazolinMeropenemCotrimoxazoleDiameter of inhibition zone (mm)30-4029-3521-2728-3423-29Table 2. Antibiotic susceptibility test on bacterial strains for three companySensitive (mm)≥21≥26≥23≥23≥16Intermediate16-2023-2520-2220-2211-15Resistant≤15≤22≤19≤19≤10AntibioticsCiprofloxacinCefotaximeCefazolinMeropenemCotrimoxazoleTable 3. Kappa values for antibiotic disks on two agar plates   Quicolor plates Mueller-Hinton agar platesSXT0.960.930.96MEM0.310.310.48CZ0.780.750.86CTX0.940.940.97CIP0.920.940.97SXT0.970.971MEM0.660.661CZ111CTX0.970.971CIP111                        DisksMast vs. Padtan TebRoscovs. Padtan TebRoscovs. MastTable 4. Percentage of agreement fortested antibiotic disks on Quicolor platesCompared groupsMast vs. Padtan TebRosco vs. Padtan TebRosco vs. MastCIP100%100%100%CTX98%98%100%CZ100%100%100%MEM98%98%100%SXT98%98%100%Table 5. Percentage of agreement for tested antibiotic disks on Mueller-Hinton agar platesCompared groupsMast vs. Padtan TebRosco vs.  Padtan TebRosco vs. MastCIP95%97%98%CTX97%97%98%CZ88%87%92%MEM94%94%97%SXT98%97%98%http://ijm.tums.ac.irNeDA SAffARI eT AL .                                                                                                            310           IRAN. J. MICROBIOL.  Volume 8 Number 5 (October 2016) 307-311 DISCUSSION    According to the results of the present study Qui-color medium can be used for rapid antibiotic suscep-tibility testing of Enterobacteriaceae in combination with standard disk diffusion method especially when urgent results are needed. Comparative statistical analysis indicated good to excellent agreements on both Quicolor and Mueller-Hinton agar plates when disks from different resources were used. CV values were satisfactory for the majority of cases using three types of disks on both media.    In the case of Quicolor medium, it should be men-tioned that the plates should be stored in refrigera-tor at 4-8°C (15, 16). In the case of longer storage, plates should be kept in dark. Development of in-hibition zones will take longer times if the depth of the agar is more than usual. Color of the agar me-dium is dark red at higher pH values and it takes a long time for developing yellow inhibition zones. Quicolor medium represents the results within 4 to 6 hours depending on special factors such as type of the strain. If diameter of the inhibition zone is not read within 8 hours, the zones will be disappeared. There are some observed unusual zones such as binary zones, zones with feather margins and some colonies within the zones which will be developed when anti-biotic disks have been located very close to each other (17).   Quality of antibiotic disks from different manufac-turers depends on several factors including quality of applied antibiotics, quality of paper or tablet disks which should be standardized (18, 19). Disks may oc-casionally contain antibacterial substances other than, or in addition to those quoted on the label (18) and the frequency of its occurrence is very difficult to es-timate. Such disks would be rarely detected through inclusion of controls in disk sensitivity tests and po-tentially are more dangerous than non-reacting disks because they can report a resistant microorganism as a sensitive one. Humidity and temperature affect the stability of an-tibiotics in disks (20-22). Consequently, disks should be stored in sealed containers, preferably containing a desiccant, at 4°C or below and should be allowed to warm up to room temperature before use.   On the other hand, in high pH values penetration of antibiotics increases which yield to larger inhibi-tion zones. Thus, all the manufacturers should keep cold production chains (5, 13, 23, 24) indicating the importance of source and origin of antibiotic disks we use.CONCLUSION   Commercially available disks are often designed for use in testing methods within the home country but they may be imported into the other countries where different methods are being used. Thus, it is important to know whether they can cause different interpreta-tions in sensitivity tests. In order to obtain reliable in-terpretations, it is necessary for each country to have its national standard quality control laboratories.ACKNOWLeDGMeNT   The authors thank vice-chancellor of Alzahra Uni-versity, Tehran, Iran, for the financial support. We also thank the staff at Department of Microbiology, Pas-teur Institute of Iran, Tehran, for providing bacterial isolates.RefeReNCeS1. Cantón R, Loza E, Del Carmen Conejo M, Baquero F, Martínez-Martínez L, MENSURA Collaborative Group. Quality control for β-lactam susceptibility test-ing with a well-defined collection of Enterobacteria-ceae and Pseudomonas aeruginosa strains in Spain. J Clin Microbiol 2003;41:1912-1918.2. Bauer AW, Kirby WM, Sherris JC, turck, Turck M. An-tibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-496.3. CLSI. Performance standards for antimicrobial suscep-tibility testing; twenty-third informational supplement. CLSI document M100-S23. Wayne, PA: Clinical and Laboratory Standard Institute; 2013.4. Brown DF, Kothari D. Comparison of antibiotic discs from different sources. J Clin Pathol 1975;28:779-783.5. Wright W. FDA actions on antibiotic susceptibility discs. In: Current techniques for antibiotic susceptibil-ity testing 1974. Charles C Thomas, Publisher, Spring-field, Ill. pp. 26-46.6. King A, Brown DF. Quality assurance of antimicrobial susceptibility testing by disc diffusion. J Antimicrob Chemother 2001;48:71-76.7. Lalitha MK. Manual on antimicrobial susceptibil-ity testing. Guide lines for antimicrobial suscep-http://ijm.tums.ac.irCOMpARISON Of DffeReNT ANTIBIOTIC DISKStibility testing.: twelfth informational supplement 2004;56238:454-456.8. Miles RS, Amyes SG. Laboratory control of antimicro-bial therapy. In: Practical Medical Microbiology 1996. Eds, Mackie and McCartney. 14th ed.New York: Chur-chill Livingstone; pp. 151-178.9. Brown DF, Kothari D. Comparison of tablets and pa-per discs for antibiotic sensitivity testing. J Clin Pathol 1975;28:983-988.10. Grove DC, Randall WA. Assay methods of antibiotics. Medical Encyclopedia, N.Y. 1955.11. O'Grady F, Lambert HP, Finch RG, Greenwood D. An-tibiotic and chemotherapy: anti-infective agents and their use in therapy. Epidemiol infect 1997. 7th edition. Eds. Pp. 987. Churchill Livingstone . ISBN 0 4340 5255 7.12. Lorian V (2005). Antibiotics in laboratory medicine. 5th ed. Techbooks, Edwards brothers.13. Piddock LJ. Techniques used for the determination of antimicrobial resistance and sensitivity in bacteria. Antimicrobial agents research group. J Appl Bacteriol 1990;68:307-318.14. Bauer W, Perry M; Kirby MM.Single-Disk Antibiot-ic-Sensitivity Testing of Staphylococci. AMA Arch In-tern Med 1959;104:208-216. 15. Kocagöz S, Budak F, Gür D. Evaluation of a chromo-genic medium for rapid detection of extended spectrum beta-lactamase producing Salmonella spp. Indian J Med Res 2006;124:443-446.16. Kocagöz T, Ercis S, Darka Ö, Salmanzadeh-Ahrabi S, Kocagöz S, Hasçelik G. Quicolor: A novel system for rapid antibacterial susceptibility testing. Ann Microbi-ol 2007;57:131-135.17. Amsterdam D (2014). Antibiotics in laboratory med-icine. 6th ed. Lippincott Williams & Wilkins Publica-tion.18. Brown DF, Selkon JB. Letter: Antibiotic discs active against resistant organisms. Br Med J 1974;1:573.19. Drew WL, Barry AL, O'Toole R, Sherris JC. Reliabil-ity of the Kirby-Bauer disc diffusion method for de-tecting methicillin-resistant strains of Staphylococcus aureus. Appl Microbiol 1972;24:240-247.20. Ericsson HM, Sherris JC. Antibiotic sensitivity test-ing. Report of an international collaborative study. Acta Pathol Microbiol Scand B Microbiol Immunol 1971;217: 1+.21. Garrod LP, Lambert HP, O'grady F (1973). General principles of treatment. In: Antibioticsand Chemother-apy. 4th ed. Edinburgh and London: Churchill Living-stone, pp. 280-281.22. Griffith LJ, Mullins CG. Drug resistance as influ-enced by inactivated sensitivity discs. Appl Microbiol 1968;16:656-658.23. Hoo R, Drew WL. Potential unreliability of nitrofuran-toin disks in susceptibility testing. Antimicrob Agents Chemother 1974;5:607-610.24. Ostrander WE, Griffith LJ. An evaluation of different types of paper concerning their suitability for sensitiv-ity discs. Antibiot Annu 1958-1959;6:813-817.IRAN. J. MICROBIOL.  Volume 8 Number 5 (October 2016) 307-311               311 http://ijm.tums.ac.ir

A comparison of antibiotic disks from different sources on Quicolor and Mueller-Hinton agar media in evaluation of antibacterial susceptibility testing

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A comparison of antibiotic disks from different sources on Quicolor and Mueller-Hinton agar media in evaluation of antibacterial susceptibility testing

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