a Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre; b Service de Bactériologie-Virologie, Centre Hospitalier et Universitaire de Rennes 35033 Rennes, France
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Abstract |
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Introduction |
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Materials and methods |
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S. flexneri strain 112540 was isolated in 1995 at the hospital Laennec (Paris, France) from a stool sample of a 16 month- old Algerian child admitted to the intensive care ward with acute and bloody diarrhoea. The isolate was identified with the API-20E system (bioMérieux, Marcy l'Étoile, France) and by slide agglutination testing with a specific antiserum (Sanofi-Diagnostics Pasteur, Marnes-La-Coquette, France). A nalidixic acid-resistant Escherichia coli HB101 strain was used as the recipient strain in mating and transformation experiments. Plasmid pBK-CMV (Stratagene, Amsterdam, The Netherlands) was used in cloning experiments. E. coli NCTC 50192 harbouring plasmids of 7, 38, 66 and 154 kb was used as a standard for plasmid size analysis.
Susceptibility testing
Disc diffusion tests were performed and interpreted according to the 1999 guidelines of the French Society for Microbiology (http://www.sfm.asso.fr/Sect4/atbfr.html). The MICs of amoxycillin, ticarcillin, piperacillin, cephalothin, cefotaxime, ceftazidime, aztreonam, cefepime, cefoxitin and imipenem were determined on MuellerHinton agar plates containing serial dilutions of appropriate antibiotics as described previously.7 Clavulanic acid and tazobactam were used at fixed concentrations of 2 and 4 mg/L, respectively.
Mating experiments and plasmid content
Direct transfer of the ß-lactam resistance marker of S. flexneri 112540 to nalidixic acid-resistant E. coli HB101 was attempted by solid mating assays at 37°C.7 Transconjugants were selected on CHROMagar-Orientation plates (CHROMagar Microbiology, Paris, France) supplemented with nalidixic acid (50 mg/L) and ceftazidime (2 mg/L) or gentamicin (50 mg/L). E. coli transconjugants (pink colonies) were differentiated from spontaneous S. flexneri mutants (white colonies). Frequency of transfer was calculated as the ratio of total number of transconjugants divided by the number of recipients. Plasmid DNA was extracted from S. flexneri 112540 and transconjugant E. coli HB101 using a Qiagen Maxi kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions.
DNA techniques
Polymerase chain reaction (PCR) experiments, endonuclease digestions, ligation, electroporation, transformation, molecular cloning and agarose gel electrophoresis were performed as described previously.7 Primers TEM-F (5'-GTATCCGCTCATGAGACAATA-3'), TEM-B (5'-TCTAAAGTATATATGAGTAAACTTGGTCTG-3'), SHV-F (5'-AAGATCCACTATCGCCAGCAGG-3') and SHV-B (5'-ATTCAGTTCCGTTTCCCAGCGG-3') were used to amplify known ß-lactamase genes (blaTEM, blaSHV) in S. flexneri 112540 and in E. coli HB101 transconjugants. BamHI-digested plasmid DNA from an E. coli transconjugant was cloned into BamHI-digested pBK-CMV phagemid and electroporated into E. coli HB101. Recombinant bacteria were plated on to trypticase soy plates containing cefotaxime (1 mg/L). PCR products and cloned fragments were sequenced on both strands using laboratory-designed primers on a sequencer (ABI 377; P. E. Biosystems, Les Ulis, France). Sequence analyses were performed online at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The published sequences will appear under Genbank accession number AF282921.
Isoelectric focusing of ß-lactamases
Supernatants of clinical S. flexneri 112540 and E. coli transconjugant sonicates were subjected to isoelectric focusing (IEF) on an ampholine polyacrylamide gel for 2 h at 10 W constant power on a flatbed apparatus (FBE-3000; Amersham Pharmacia Biotech, Orsay, France) as described previously.7 The ß-lactamases were visualized with an overlay of nitrocefin (0.2 g/L). The pIs were determined by comparison with those of known ß-lactamases.
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Results and discussion |
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IEF experiments with S. flexneri 112540 and the E. coli transconjugant showed two ß-lactamases with pIs of 5.4 and 7.6, similar to those of TEM-1 and SHV-1, respectively. The corresponding ß-lactamase genes were amplified from plasmid pSF-1 from the transconjugant, yielding a 956 bp and a 231 bp fragment with TEM- and SHV-specific primers, respectively. Direct DNA sequencing of the TEM PCR product revealed identity with the entire blaTEM-1B gene identified in S. flexneri strain K24.8 Direct sequencing of the 231 bp SHV PCR product revealed identity with several partial blaSHV sequences. In order to obtain the entire blaSHV sequence along with flanking sequences, plasmid pSF-1 was digested with BamHI and cloned into pBK-CMV. All the recombinant plasmids conferring resistance to cefotaxime contained a 3.5 kb insert. One of them, pSF-2, contained a 861 bp open reading frame (ORF) identical to that of blaSHV-2 (Figure).9 The extended spectrum of activity to oxyimino-cephalosporins and aztreonam of SHV-2 is caused by a single amino acid substitution (glycine at position 238 in SHV-1 is replaced by serine in SHV-2). The recombinant E. coli strain harbouring pSF-2 was less susceptible to extended-spectrum cephalosporins and aztreonam, with higher MICs than those of S. flexneri 112540 and the transconjugant E. coli HB101 (pSF-1) strain (Table
). The high copy number of pBK-CMV (200400 copies/cell) may explain this discrepancy.
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This is the first report of a clinical isolate of Shigella spp. producing an ESBL. Recently, SHV-11, a point mutation derivative of SHV-1 (leucine to glutamine replacement at position 35),3 was detected in a clinical isolate of S. dysenteriae in India.4 However, this enzyme confers a resistance phenotype indistinguishable from that of SHV-1 and therefore cannot be considered as an ESBL.3
The emergence of drug-resistant Shigella spp. is of particular concern even in developed nations.1 Appropriate antimicrobial treatment of shigellosis may limit the clinical course of illness and the duration of faecal excretion of bacteria. Although fluoroquinolones remain a valuable choice for treatment in adults, they are not recommended for children. Infections by multidrug-resistant, ESBL-producing shigella strains spreading among the community or leading to nosocomial outbreaks could be a therapeutic challenge in the future.
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Acknowledgments |
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Notes |
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References |
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2
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8 . Siu, L. K., Ho, P. L., Yuen, K. Y., Wong, S. S. & Chau, P. Y. (1997). Transferable hyperproduction of TEM-1 ß-lactamase in Shigella flexneri due to a point mutation in the Pribnow box. Antimicrobial Agents and Chemotherapy 41, 46870.[Abstract]
9 . Podbielski, A. & Melzer, B. (1990). Nucleotide sequence of the gene encoding the SHV-2 ß-lactamase (blaSHV-2) of Klebsiella ozaenae. Nucleic Acids Research 18, 4916.[ISI][Medline]
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Blattner, F. R., Plunkett, G., Bloch, C. A., Perna, N. T., Burland, V., Riley, M. et al. (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 145374.
Received 30 August 2000; returned 18 October 2000; revised 10 November 2000; accepted 29 November 2000