Antimicrobial susceptibility testing of Salmonella spp.,
Campylobacter spp., indicator E. coli, Enterococcus
spp. and the veterinary pathogens was performed as microbroth dilution MIC with the Sensititre system (Trek Diagnostic Systems Ltd., UK). Inoculation and incubation procedures were in accordance with the CLSI guidelines. The following quality control strains were used for internal control: Staphylococcus
aureus ATCC 29213, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterococcus faecalis ATCC 29212 and Campylobacter jejuni ATCC
33560.
An overview of the interpretation of MIC-values is presented in Table 56. Since 2007, data were interpreted using EUCAST epidemiological cut-off values, and if not available, EUCAST or CLSI clinical breakpoints were applied. Exceptions and further details are described in Table 56. Data from previous years presented in this DANMAP report are not corrected for the change in interpretation (e.g. all data are presented with use of the interpretation applied for the year in question). All MIC-distributions for human isolates are presented with both epidemiological cut-off values and clinical breakpoints in order to visualize the impact of changing the interpretation criteria.
All isolates from animals and foods were susceptibility tested at the National Food Institute, DTU, except for broilers, where the testing was performed at the National Veterinary Institute, DTU. The Salmonella spp. and Campylobacter spp. of human origin were susceptibility tested at the SSI.
From 1998–2007, a performance test for susceptibility testing was carried out almost once a year to ascertain the quality and comparability of susceptibility testing in the laboratories providing MIC-data. Today, the laboratories are accreditated by DANAK (the Danish national body for accreditation) or awaiting an accreditation, ensuring MIC-data of high quality. One isolate per bacterial species per herd, per food sample, or per patient was tested for antimicrobial susceptibility. For animal isolates in excess numbers (indicator E.coli, enterococci and Campylobacter spp. from healthy production animals), a random selection of 100 or 150 isolates was appointed for susceptibility testing. Due to a relatively low number of isolates, C.
jejuni from pigs and C. coli from cattle and broilers were
not susceptibility tested.
Staphylococcus aureus from humans
Susceptibility testing was performed using the tablet diffusion method (Neo-Sensitabs®, A/S Rosco, Denmark) on Danish Blood Agar (SSI Diagnostika, Denmark) towards penicillin, cefoxitin, streptomycin, kanamycin, erythromycin, clindamycin (only when an isolate was resistant to erythromycin), tetracycline, fusidic acid, rifampicin, norfloxacin, mupirocin and linezolid. A cefoxitin 60 µg tablet was used for screening for methicillin susceptibility. Isolates with an inhibition zone <29 mm were further tested for the presence of the mecA gene by PCR. In addition, MRSA isolates were screened for susceptibility towards glycopeptides by spot test on Brain-Heart infusion (BHI) agar (Becton Dickinson, Germany) with teicoplanin (5 mg/L) and confirmed using Etest® (AB Biodisk, Sweden) on BHI with inoculum of McFarland 2.0. In case of MIC ≥8 mg/L for vancomycin and teicoplanin or an MIC ≥12 mg/L for teicoplanin, population analysis profile against vancomycin was performed [Wootton et
al. 2001. J. Antimicrob. Chemother. 47: 399-403]. Invasive Streptococcus pneumonia from humans
Screening for penicillin-resistant S. pneumoniae was performed using a 1 μg oxacillin tablet (Neo- Sensitabs®, A/S Rosco, Taastrup, Denmark) on 10% horse blood agar (SSI Diagnostika, Hillerød, Denmark),
and for erythromycin-resistant S. pneumoniae using a 78 μg erythromycin tablet (Neo-Sensitabs®, A/S
Rosco, Taastrup, Denmark) on 10% horse blood agar (SSI Diagnostika). The breakpoints used are those defined by the CLSI. Penicillin and erythromycin MICs are determined using the Etest (AB Biodisk, Solna, Sweden) on Danish Blood Agar (Resistensplade, SSI Diagnostika) incubated at 36°C, 5% CO2. The
breakpoints used are those defined by Etest.
Invasive Streptococcus pyogenes (group A), group B, C and G streptococci from humans Screening for penicillin-resistant streptococci was performed using a 1 μg oxacillin disk (Oxoid, Greve, Denmark) on 10% horse blood agar (SSI Diagnostika, Hillerød, Denmark), and for erythromycin-resistant streptococci using a 78 μg erythromycin tablet (Neo- Sensitabs®, A/S Rosco, Taastrup, Denmark) on 10%
horse blood agar (SSI Diagnostika). Erythromycin
resistant streptococci are tested with 15 μg erythromycin disk (Oxoid) and 15 μg clindamycin disk (Oxoid, Greve, Denmark) on Danish Blood Agar (Resistensplade, SSI Diagnostika). Erythromycin MICs are determined using the Etest (AB Biodisk, Solna, Sweden) on Danish Blood Agar (Resistensplade, SSI Diagnostika) incubated at 36°C, 5% CO2. The breakpoints used are those defined by the CLSI. Resistant isolates are defined as both fully and intermediary resistant isolates.
E.coli, Klebsiella pneumoniae, Pseudomo- nas aeruginosa, non-invasive Streptococcus pneumoniae, non-invasive Streptococcus pyogenes, invasive E. faecium and E. faecalis
from humans
In 2008, the DCM at Statens Serum Institut, the hospitals in Næstved, Odense and Viborg, and Rigshospitalet, which is the national referral hospital,
Table 56. Interpretation used for MIC-determination on bacterial isolates from animals, food and humans.
Epidemiological cut-off values and clinical breakpoints recommended by EUCAST are marked in grey.
EUCAST epidemiological cut-off values were used for interpretation. If not available, interpretation criteria were applied by DANMAP. a) Trade name synercid. EUCAST epid. cut-off value (>1) was not applied according to investigations presented in DANMAP 2006. b) CLSI clinical breakpoint was applied.
* CLSI clinical breakpoint (presented if EUCAST clinical breakpoints are not available).
Antimicrobial agent Salmonella E. coli faeciumE. E. faecalis C. jejuni C. coli Epid Clin Epid Clin Epid Clin Epid Clin Epid Clin Epid Clin cut-off break cut-off break cut-off break cut-off break cut-off break cut-off break
mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml Ampicillin >4 >8 >8 >8 >4 >8 >4 >8 Apramycin >16 >16 Avilamycin >16 >8 Cefotaxime >0.5 >2 >0.25 >2 Cefoxitin Ceftiofur >2 >1 Chloramphenicol >16 >16* >16 >16* >32 >16* >32 >16* >16 >16 Ciprofloxacin >0.06 >1 >0.03 >1 >1 >1 >1 >1 Colistin >2 >2 >2 >2 Erythromycin >4 >4* >4 >4* >4 >4 >16 >16* Florfenicol >16 >16 Gentamicin >2 >4 >2 >4 >32 >512* >32 >512* >1 >2 Kanamycin >1,024 >1,024 Linezolid >4 >4 >4 >4 Nalidixic acid >16 >16* >16 >16* >16 >32 Neomycin >4 >8 Penicillin >16 >8* >16 >8* Quinupristin/ dalfopristin a) >4 b) >4 Salinomycin >4 >4 Spectinomycin >64 >64 Streptomycin >16 >16 >128 >512 >2 >4 Sulfonamide >256 b) >256* >256 b) >256* Tetracycline >8 >8* >8 >8* >4 >8* >4 >8* >2 >8* >2 >8* Tiamulin Tigecycline >0.25 >0.5 >0.25 >0.5 Trimethoprim >2 >4 >2 >4 Vancomycin >4 >4 >4 >4 DANMAP 2009
used the tablet diffusion method (Neo-Sensitabs®,
A/S Rosco) on Danish Blood Agar (Resistensplade, SSI Diagnostika) and the breakpoints defined for this medium by A/S Rosco.
However, the DCM at Odense Hospital used Neo- Sensitabs® on Müeller-Hinton II agar (SSI Diagnostica)
when testing urine isolates and Columbia agar with 4.5% NaCl (SSI Diagnostika) for oxacillin-susceptibility of staphylococci. The DCM at Vejle Hospital used the Neo-Sensitabs® on Müeller-Hinton II agar (SSI
Diagnostica) and the breakpoints defined for this medium by A/S Rosco. The DCM at Esbjerg Hospital used the tablet diffusion method (Neo-Sensitabs®, A/S
Rosco) on Müeller-Hinton II agar (SSI Diagnostika) when testing E. coli. The DCM at Aalborg Hospital also used the Neo-Sensitabs® on Mueller-Hinton II agar (SSI
Diagnostika) in combination with the tablet diffusion method (A/S Rosco) and the breakpoints defined by the Swedish Reference Group for Antibiotics (SRGA). The only material exception from SRGA was that the wildtype population of E. coli was deemed susceptible for ampicillin (and not intermediary susceptible). In 2008, the DCM at Hillerød, Hvidovre, Herlev, Herning and Århus Hospitals used the disk diffusion method (Oxoid, Basingstoke, UK) on Iso-Sensitest (ISA) medium (Oxoid). The DCM at Slagelse Hospital used the same disks on Iso-Sensitest (ISA) medium with or without 5% horse blood (Oxoid) according to test material and bacterial species. All laboratories performing the disk diffusion method used the breakpoints defined by the Swedish Reference Group for Antibiotics (Available from: URL: http://www.srga. org/). However, the DCM at Århus Hospital changed to EUCAST breakpoints for urine samples continuously during 2009 and for enterobacteria in blood per November 2009.
All submitting laboratories participate in national and international quality assurance collaborations such as the United Kingdom National External Quality Assessment Schemes (NEQAS).
Quinupristin/dalfopristin breakpoint The epidemiological cut-off value suggested by EUCAST for quinupristin/dalfopristin when testing
E. faecium is >1 μg/ml. In DANMAP, E. faecium
isolates with MICs >4 μg/ml are reported resistant to quinupristin/dalfopristin due to an evaluation study presented in the DANMAP 2006 report, page 49-50.