EUCAST posters at ECCMID 2012

66668888066668888P682EUCAST standardised disk diffusion methodologyfor Campylobacter jejuni and C. coliE. Matuschek 1 , K. Veldman 2 , A. Hakanen 3 , S. Bengtsson 1 , M. Lehtopolku 3 , D. Mevius 2 and G. Kahlmeter 11EUCAST Laboratory for AST, Växjö, Sweden, 2 Central Veterinary Institute (CVI), Lelystad, the Netherlands3National Institute for Health and Welfare (THL), Turku, FinlandIntroductionAntimicrobial susceptibility testing of Campylobacter spp. inclinical laboratories is often performed using gradient MIC testsor disk diffusion, but standardised methodology and interpretivecriteria are often lacking. The European Committee onAntimicrobial Susceptibility Testing (EUCAST) is in the processof establishing clinical MIC breakpoints for C. jejuni and C. coli.ObjectiveThe objective of this study was to investigate if the standardEUCAST MH-F medium and disk diffusion criteria can be usedfor reproducible antimicrobial susceptibility testing of C. jejuniand C. coli.MethodsPreliminary testing was performed on 15 C. jejuni and 15 C. colifrom the Netherlands with known MIC values (brothmicrodilution) including isolates with and without resistancemechanisms. Additional testing was performed on 17 C. jejuniand 12 C. coli from Finland. All isolates were tested in triplicatefor ciprofloxacin 5 µg, erythromycin 15 µg and tetracycline 30 µg.Investigated variables are shown in Table 1.Table 1Investigated variablesMediumIncubationReading of zonesResultsUnsupplemented Mueller-Hinton agar (MH) and Mueller-Hinton agarwith 5% defibrinated horse blood and 20 mg/L β-NAD (MH-F)35, 37, 39 and 41°C24 and 44 hSmallest and largest appearing zone when tilting the plateAll tests were performed with a 0.5 McFarland inoculum and microaerobicincubation in plastic jars.The proposed EUCAST disk diffusion methodology for C. jejuniand C. coli is presented in Table 2. BothC. jejuni and C. coligrew best on MH-F plates at 41°C. All C. jejuni grew well after 24h of incubation, whereas some C. coli required 44 h beforeinhibition zones could be read. Swarming was avoided by dryingplates before inoculation.All isolates with MICs above the epidemiological cut off (ECOFF)were identified as non-wild type also with the disk diffusion test(Figure 1), irrespective of whether the smallest or largest zonewas used. The results were reproducible within and between thesites. The standard deviation for triplicate tests ranged from 0.4-1.7 mm, with the largest variation for inhibition zones read after44 h of incubation. Examples of combined inhibition zonedistributions from several sites are shown in Figure 2.No of isolatesNo of isolatesNo of isolatesNo of isolatesReproducible disk diffusion testing of C. jejuni and C. coli can beperformed using the EUCAST MH-F media. Swarming waseffectively avoided by pre-drying of plates. Disk diffusioncorrelated well to MIC determination and reliably distinguishedbetween wild-type and non-wild type isolates. Further evaluationof the methodology is currently performed at several laboratoriesin Europe.40353025201510180160140120100807060504030201050121086420806040200ConclusionsTable 2EUCAST proposed disk diffusion methodology for Campylobacter jejuni andCampylobacter coli .Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/L β-NAD (MH-F)Medium(Plates should be dried before incubation to avoid swarming, either atroom temperature (20-22°C) over night or at 35°C for 1-2 hours.)Inoculum McFarland 0.5IncubationReading of zonesCiprofloxacin 5 µg vs. MIC, Campylobacter jejuni and coli57 clinical isolates tested in duplicate1010ECOFF: WT ≤ 1 mg/L1012Campylobacter jejuni with ciprofloxacin 5 µg414 clinical isolates121412141614161816182022182024202226Erythromycin 15 µg vs. MIC, Campylobacter jejuni30 clinical isolates tested in duplicateECOFF: WT ≤ 4 mg/L2422282628303234Inhibition zone diameter (mm)Campylobacter coli with ciprofloxacin 5 µg170 clinical isolates3024322634Inhibition zone diameter (mm)28Inhibition zone diameter (mm)Microaerobic environment41°C24 hours ± 30 minutes(C. coli with non-sufficient growth after 24 h incubation are reincubatedimmediatly and read after totally 40-48 h incubation.)EUCAST standard reading instructions:Read MH-F plates from the front with the lid removed illuminated withreflected light. Zone edges should be read at the point of completeinhbition as judged by the unaided eye.3630383640323842344044364642483844504046MIC(mg/L)≥32481684210.50.25≤0.12MIC(mg/L)≥12864321684210.550No of isolatesNo of isolatesNo of isolatesNo of isolates1098765432103530252015105050454035302520151050605040302010Tetracycline 30 µg vs. MIC, Campylobacter jejuni and coli29 clinical isolates tested in duplicate1010ECOFF: WT ≤ 2 mg/LErythromycin 15 µg vs. MIC, Campylobacter coli27 clinical isolates tested in duplicate1012Campylobacter jejuni with tetracycline 30 µg409 clinical isolates12141214161416101214161820222426283032343638404244184616481850202218202420222624222826Campylobacter coli with tetracycline 30 µg168 clinical isolates24302832263034Inhibition zone diameter (mm)ECOFF: WT ≤ 16 mg/L28Inhibition zone diameter (mm)Figure 1. Inhibition zone distributions for C.jejuni and C. coli with MIC values as colouredbars. Inhibition zones were read according to EUCAST standard reading instructions at onesite on MH from two manufacturers. Data for erythromycin are presented separately, due todifferent ECOFFs.3236303438Inhibition zone diameter (mm)364038404244464850010121416182022242628303234363840424446485032423444364638485040MIC(mg/L)≥12864321684210.50.250.12MIC(mg/L)≥12832168421Inhibition zone diameter (mm)Inhibition zone diameter (mm)For more information, please contact:erika.matuschek@ltkronoberg.seFigure 2. Combined inhibition zone distributions for ciprofloxacin and tetracycline from 4-6 sites for C. jejuni and C. coli, respectively.