Escherichia coli of animal origin in Norway contains a blaTEM-20-carrying plasmid closely related to blaTEM-20 and blaTEM-52 plasmids from other European countries.

Sir, The situation regarding antimicrobial resistance in bacteria from food-producing animals in Norway is, in an international perspective, favourable. The resistance frequencies are moderate and the situation has been stable since the start of the Norwegian monitoring programme in the veterinary sector (NORM-VET) (www. vetinst.no) in the year 2000. We report the first bacterial isolate of animal origin detected in Norway with reduced susceptibility to cephalosporins. The isolate (Escherichia coli 2006-01-1248, hereafter termed E. coli 1248) originated from a broiler domesticated in a cephalosporin-free environment and was included in the NORM-VET 2006 programme. MICs of cefotaxime, ceftiofur and ampicillin were 1, 4 and .32 mg/L, respectively. The cefotaxime MIC of 1 mg/L is close to the clinical breakpoint recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (susceptible 1 mg/L, resistant .2 mg/L). However, this value is considerably higher than those observed for susceptible E. coli strains (wild-type population) having MICs of 0.25 mg/L (www.eucast.org). MICs of cefalotin, ceftazidime and cefepime, determined using Etest (AB Biodisk, Solna, Sweden) with E. coli ATCC 25922 as susceptible control, were 64, 0.5 and 0.25 mg/L, respectively. E. coli 1248 was positive in the double-disc synergy test and in the confirmatory test. The tests were carried out as recommended by the manufacturer using discs containing cefotaxime (30 mg), ceftriaxone (30 mg), ceftazidime (30 mg), cefepime (30 mg), amoxicillin/clavulanic acid (30 mg/15 mg), and ceftazidime and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark) (User’s guide NEO-SENSITABS susceptibility testing 19th Edn, www.rosco.dk). PCR was performed for the detection of blaCTX-M, blaSHV and blaTEM 1,2 using the following control strains: E. coli K4-23 (blaCTX-M-9), E. coli Dak2 (blaSHV) and E. coli 76-33094-7 (blaTEM). Amplicons were produced with the blaTEM-specific primers only. Sequencing showed that a blaTEM-20 gene variant 3 was present. Conjugation showed that blaTEM-20 was located on a self-transferable plasmid. The transconjugant had the following MICs of b-lactams: ampicillin, .32 mg/L; cefotaxime, 0.5 mg/L; and ceftiofur, 1 mg/L. The blaTEM-20 sequence contained three silent mutations at positions 346, 682 and 925 when compared with a previously published sequence (accession number Y17581). The other blaTEM-20 gene variant, conferring high-level resistance to thirdgeneration cephalosporins, showed a 135 bp deletion in the promoter and a G!T mutation at position 162. These mutations were not identified in the sequence from E. coli 1248, and this may explain the lower MICs exhibited. The blaTEM-20 gene has previously been detected in Salmonella Paratyphi B dTþ from poultry in the Netherlands. The strain Salmonella Paratyphi B dTþ 63.48, kindly donated to us for further investigation, was compared with E. coli 1248. The blaTEM-20 gene was also carried by a conjugative plasmid in the Salmonella Paratyphi B dTþ strain, and the two blaTEM-20 sequences were 100% identical. The transconjugant was resistant to b-lactams only with the same MICs as the E. coli 1248 transconjugant. Both blaTEM-20 plasmids were assigned to the incompatibility (Inc) group I1 by PCR-based replicon typing. Plasmid multilocus sequence typing (pMLST) for subtyping IncI1 plasmids was applied to the blaTEM-20 plasmids. 5 The following alleles (accession numbers) were obtained: repI1-1 (EU370458), ardA-2 (EU370453), trbA-pndC-2 (EU40466), sogS-3 (EU70463) and pilL-3 (EU370457). These alleles corresponded to sequence type 5, previously assigned to an IncI1 plasmid carrying blaTEM-52 identified in a Salmonella Infantis strain isolated in Belgium in 2005 (the alleles showed 100% nucleotide identity except for one nucleotide in the ardA locus). This blaTEM-52 plasmid is reported to be widely disseminated among different Salmonella serovars from poultry and humans in Belgium and France. The blaTEM-20 plasmids were large (.100 kb) and showed profiles that seemed to be similar when comparing the PstI and EcoRI restriction patterns with published restrictions patterns of the blaTEM-52 plasmid. 5–7