JAC Advance Access originally published online on October 29, 2008
Journal of Antimicrobial Chemotherapy 2009 63(1):215-216; doi:10.1093/jac/dkn445
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Research letters |
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
1 Section of Bacteriology, National Veterinary Institute, Oslo, Norway 2 The Norwegian Zoonosis Centre, National Veterinary Institute, Oslo, Norway 3 Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Rome, Italy 4 The Norwegian School of Veterinary Science, Department of Production Animal Clinical Sciences, Sandnes, Norway
* Corresponding author. Tel: +47-23-21-63-81; Fax: +47-23-21-63-01; E-mail: marianne.sunde{at}vetinst.no
Keywords: E. coli , extended-spectrum β-lactamases , ESBLs , IncI1 plasmids , broiler , pMLST
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 µg), ceftriaxone (30 µg), ceftazidime (30 µg), cefepime (30 µg), amoxicillin/clavulanic acid (30 µg/15 µg), and ceftazidime and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark) (User’s guide NEO-SENSITABSTM susceptibility testing 19th Edn, www.rosco.dk).
PCR was performed for the detection of blaCTX-M, blaSHV and blaTEM1,2 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 variant3 was present. Conjugation showed that blaTEM-20 was located on a self-transferable plasmid. The transconjugant had the following MICs of β-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
[GenBank]
).3 The other blaTEM-20 gene variant, conferring high-level resistance to third-generation 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 dT+ from poultry in the Netherlands.2 The strain Salmonella Paratyphi B dT+ 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 dT+ strain, and the two blaTEM-20 sequences were 100% identical. The transconjugant was resistant to β-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.4
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 [GenBank] ), ardA-2 (EU370453 [GenBank] ), trbA-pndC-2 (EU40466), sogS-3 (EU70463) and pilL-3 (EU370457 [GenBank] ). 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 20055 (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.6 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
The blaTEM-52 gene has been identified within a Tn3 transposon derivative on the plasmid described earlier (accession number EF141186 [GenBank] ).6 Primers were designed in order to amplify and sequence a similar region (6.6 kb) in E. coli 1248 and in Salmonella Paratyphi B dT+. A Tn3-related transposon, with 100% identical nucleotide sequence, was detected in both strains. Only one amino acid, E104K, distinguishes TEM-20 from TEM-52. Comparison between the blaTEM-20 sequence and the blaTEM-52 sequence revealed only one nucleotide difference (producing amino acid alteration E104K). Five additional nucleotide differences were found when the remaining parts of the DNA region were aligned, indicating a close relationship between the blaTEM-20 and blaTEM-52 transposons. In conclusion, two closely related plasmid scaffolds carrying identical transposons were identified, associated with blaTEM-20 in E. coli from Norway and in Salmonella Paratyphi B dT+ from the Netherlands, and with blaTEM-52 in Salmonella from Belgium and France.
Detection of an extended-spectrum β-lactamase (ESBL)-positive E. coli from livestock in Norway was unexpected as there are no commercial preparations containing cephalosporins licensed for veterinary use. An interview with the farmer confirmed that there had been no use of cephalosporins for animals or humans at the farm. The strain, or plasmid, might have been part of bacterial flora of animals taken to Norway for breeding purposes.
The blaTEM-20 plasmid conferring low-level resistance to cephalosporins may represent a challenge with regard to detection of ESBL-positive isolates. The plasmids might also have the potential to develop into variants conferring higher levels of resistance as only one mutation may contribute to a change in the phenotype.
The nucleotide sequence determined is available under accession number EU527189.
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This work was partly supported by a grant (167822/I10) from the Norwegian Research Council and partly funded by the National Veterinary Institute, Norway.
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None to declare.
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This study was presented at the Second Symposium on Antimicrobial Resistance in Animals and the Environment, Tours, France, 2007. Dik Mevius (Central Veterinary Institute, Wageningen, the Netherlands) and Henrik Hasman (Technical University of Denmark) are acknowledged for providing the Salmonella Paratyphi B dT+ strain. Arnfinn Sundsfjord (University Hospital of North Norway) and Frank M. Aarestrup (Technical University of Denmark) are acknowledged for donating PCR control strains.
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1 Brinas L, Zarazaga M, Sáenz Y, et al. β-Lactamases in ampicillin-resistant Escherichia coli isolates from foods, humans, and healthy animals. Antimicrob Agents Chemother (2002) 46:3156–63.
2
Hasman H, Mevius D, Veldman K, et al. β-Lactamases among extended-spectrum β-lactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in the Netherlands. J Antimicrob Chemother (2005) 56:115–21.
3
Arlet G, Goussard S, Courvalin P, et al. Sequences of the genes for the TEM-20, TEM-21, TEM-22 and TEM-29 extended-spectrum β-lactamases. Antimicrob Agents Chemother (1999) 43:969–71.
4 Carattoli A, Bertini A, Villa L, et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods (2005) 63:219–28.[CrossRef][Web of Science][Medline]
5
García-Fernández A, Chiaretto G, Bertini A, et al. Multilocus sequence typing of IncI1 plasmids carrying extended-spectrum β-lactamases in Escherichia coli and Salmonella of human and animal origin. J Antimicrob Chemother (2008) 61:1229–33.
6
Cloeckaert A, Praud K, Doublet B, et al. Dissemination of an extended-spectrum-β-lactamase blaTEM-52 gene-carrying IncI1 plasmid in various Salmonella enterica serovars isolated from poultry and humans in Belgium and France between 2001 and 2005. Antimicrob Agents Chemother (2007) 51:1872–5.
7
Weill FX, Demartin M, Fabre L, et al. Extended-spectrum-β-lactamase (TEM-52)-producing strains of Salmonella enterica of various serotypes isolated in France. J Clin Microbiol (2004) 42:3359–62.
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