JAC Advance Access originally published online on May 18, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 1113-1114
© 2004 The British Society for Antimicrobial Chemotherapy
Correspondence |
Characterization of the first CTX-M-14-producing Salmonella enterica serotype Enteritidis isolate
1 Microbiology Department, University Hospital ‘Virgen Macarena’, Ave Dr Fedriani s/n, 41009 Seville, Spain; 2 Federal Institute for Risk Assessment (BfR), National Salmonella Reference Laboratory (NRL-Salm), Diedersdorfer Weg 1, 12277 Berlin, Germany
Keywords: extended-spectrum ß-lactamases, ESBLs, CTX-M
Sir,
A 10-month-old girl was admitted to the paediatric ward with feverish convulsions and pharyngeal symptoms in June 2003. During her stay, she presented diarrhoea limited to 48 h. A cefotaxime-susceptible Salmonella enterica (isolate 1) serotype Enteritidis was isolated from a blood culture sent on day 2 after admittance. She was treated with amoxicillin for 24 h and then with ceftriaxone for 5 days. Before she was discharged free of symptoms, a stool sample was sent for culture and a cefotaxime-resistant S. Enteritidis (isolate 2) was recovered. After 15 days, a faeces control culture also showed positive for a cefotaxime-resistant S. Enteritidis (isolate 3). Furthermore, a cefotaxime-resistant Escherichia coli was grown in the same control sample (isolate 4). In the two faecal samples, no signs of the cefotaxime-susceptible Salmonella were found.
S. Enteritidis isolate 1 was susceptible to all the antibiotics except nalidixic acid. S. Enteritidis isolates 2 and 3 and E. coli isolate 4 were resistant to ampicillin, piperacillin, cefotaxime and cefepime, but susceptible to ceftazidime, cefoxitin, aminoglycosides, co-trimoxazole, co-amoxiclav and piperacillin/tazobactam. The three resistant isolates 2, 3 and 4 showed synergy between clavulanic acid and third-generation cephalosporins compatible with extended spectrum ß-lactamase (ESBL). ß-Lactamases were extracted by sonication from isolates 2, 3 and 4, subjected to isoelectric focusing (IEF) gel electrophoresis in a Phast System (PhastGel IEF 3-9; Pharmacia AB, Uppsala, Sweden) and then detected using 0.5 mg/mL nitrocefin (Oxoid, Hampshire, UK). Sonicated extracts of TEM-1, TEM-24, SHV-2 and CTX-M-9 ß-lactamases were used as controls of pI. Isolates 2, 3 and 4 produced a ß-lactamase with a pI of 8.0 which suggested the presence of a CTX-M-type enzyme. For these isolates, it was possible to amplify an internal blaCTX-M fragment,1 and analysis of the deduced amino acid sequence showed that the corresponding gene encoded a CTX-M-14 ß-lactamase.
The gene blaCTX-M-14 was located on 
70 kb plasmids in S. Enteritidis isolates 2 and 3, but on an 80 kb plasmid in E. coli isolate 4. EcoRI restriction patterns showed that the two 70 kb plasmids from S. Enteritidis were indistinguishable and in part related to the 80 kb plasmid from E. coli. To determine the transfer abilities of these plasmids, conjugation experiments were carried out. When E. coli J53-AR (resistant to sodium azide) was used as the recipient strain and isolates 2, 3 and 4 served as donors, transconjugants were selected on Mueller–Hinton agar with 2 mg/L cefotaxime and 200 mg/L sodium azide. When S. Enteritidis isolate 1 served as recipient and E. coli isolate 4 as donor, transconjugants were selected on MacConkey agar with 2 mg/L cefotaxime and 50 mg/L nalidixic acid. Both types of blaCTX-M-14-carrying plasmids proved to be conjugative (Figure 1a) and harboured the blaCTX-M-14 gene on an EcoRI-fragment of 18 kb. All three S. Enteritidis isolates showed closely related XbaI-PFGE-patterns, determined as previously described.2 The only difference in the fragment patterns was an extra band of 75 kb in the cefotaxime-resistant isolates 2 and 3 (Figure 1b), which hybridized with the CTX-M-14 probe (data not shown).
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. The arrow indicates the EcoRI-fragment of Based on these observations, it was assumed that the child was infected with a strain which developed an ESBL phenotype during the treatment. Because data were not available about intestinal flora of the patient before the bacteraemic episode, we cannot exclude the possibility that the child acquired an invasive cefotaxime-susceptible and a cefotaxime-resistant S. Enteritidis as well as an ESBL-producing E. coli at the same time before hospital admission. Simultaneous transmission of ESBL-producing E. coli with susceptible Salmonella enterica from a common source has been previously reported3 in a Salmonella outbreak in a summer camp. None of the samples from campers or food-handlers yielded resistant salmonellae and, in contrast with our case, no person received antibiotic therapy.
The family of the CTX-M-type ESBLs comprises 40 enzymes, subclassified into five major groups by their similar amino acid sequences: CTX-M-1, M-2, M-8, M-9 and M-25 groups.4 Salmonella isolates have been found to express CTX-M-2, M-3, M-4 and M-9 enzymes in serotypes Typhimurium and Enteritidis isolated in South America and Europe.4 Until now, CTX-M-14 has been described in several enterobacterial species, but not in Salmonella isolates.5 In Spain, the only report of a CTX-M-type production in Salmonella was that described by Simarro et al.1 in 1997. In this study, four Salmonella enterica Virchow strains were reported with CTX-M-9 production in Barcelona and Murcia. Furthermore, CTX-M-14 ß-lactamase has previously been found in the north-west area of Spain6 in different E. coli strains, causing infections in 17 patients. Our findings provide the first evidence of CTX-M-14 ß-lactamase in Salmonella enterica serotype Enteritidis. The observation that the blaCTX-M-14 gene is located on a conjugative plasmid which can transfer in vitro between E. coli and Salmonella Enteritidis strongly suggests that such transfer events can also occur in vivo.
Acknowledgements
We thank the personnel of the NRL-Salm (BfR, Berlin), especially E. Junker and M. Jaber, for their helpful assistance. This study was supported by REIPI grant; CO3/14, Spain.
Footnotes
* Corresponding author. Tel: +34-955-008138; Fax: +34-954-377413; E-mail: Lromeroperez{at}us.es 
References
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Simarro, E., Navarro, F., Ruiz, J. et al. (2000). Salmonella enterica serovar Virchow with CTX-M-like ß-lactamase in Spain. Journal of Clinical Microbiology 38, 4676–8.
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Miko, A., Guerra, B., Schroeter, A. et al. (2002). Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar Paratyphi B isolates. Journal of Clinical Microbiology 40, 3184–91.
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Chanawong, A., M’Zali, F. H., Heritage, J. et al. (2002). Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People’s Republic of China. Antimicrobial Agents and Chemotherapy 46, 630–7.
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Bou, G., Cartelle, M., Tomas, M. et al. (2002). Identification and broad dissemination of the CTX-M-14 ß-lactamase in different Escherichia coli strains in the northwest area of Spain. Journal of Clinical Microbiology 40, 4030–6.
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