Journal of Antimicrobial Chemotherapy (2001) 47, 685-688
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
SHV-type extended-spectrum ß-lactamase in a Shigella flexneri clinical isolate
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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A Shigella flexneri isolate resistant to oxyimino-cephalosporins was recovered from a stool sample of a 16 month-old Algerian child hospitalized in Paris, France. This isolate harboured an SHV-2 ß-lactamase gene located on a c. 80 kb self-transferable plasmid. This is the first report of an Ambler class A extended-spectrum ß-lactamase from Shigella spp.
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Introduction |
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Infections by Shigella spp. are an important cause of acute diarrhoea worldwide and are responsible for 600000 deaths per year, mostly in undernourished children. Isolation of Shigella sonnei and Shigella flexneri resistant to ampicillin, tetracycline, chloramphenicol and co-trimoxazole is of growing concern,1 although most strains remain susceptible to quinolones and oxyimino-cephalosporins.1 TEM-1, OXA-1 and OXA-3 are the most frequent types of ß-lactamase reported in Shigella spp.2 Recently, SHV-11, a variant of the narrow-spectrum ß-lactamase SHV-1,3 was reported in Shigella dysenteriae.4 A class B ß-lactamase, IMP-3 (originally named MET-1), has been reported from a single S. flexneri isolate in Japan.5 Extended-spectrum ß-lactamases (ESBLs) are mainly plasmid-mediated Ambler class A enzymes, derived from SHV-1, TEM-1 or TEM-2, by one or more amino acid substitutions. Their presence can be detected by synergy between a broadspectrum cephalosporin (e.g. ceftazidime) and clavulanic acid in a double-disc diffusion test. Such enzymes, initially reported in the early 1980s in Germany and France among Klebsiella spp.,6 are often associated with strains causing nosocomial outbreaks. They have now spread worldwide and have been reported during the last decade in numerous other members of the family Enterobacteriaceae, including Salmonella spp.6 We describe in this study the first report of a multidrug-resistant Shigella isolate producing a plasmid-encoded class A ESBL.
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Materials and methods |
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Bacterial strains and plasmids
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 Mueller–Hinton 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The results in the Table
show that S. flexneri 112540 was resistant to cefotaxime and ceftazidime but susceptible to cefoxitin and that addition of clavulanic acid (2 mg/L) restored the activity of cefotaxime and ceftazidime. The double-disc diffusion test also showed synergy between ceftazidime or cefotaxime and clavulanic acid, suggesting the presence of an ESBL. According to routine antibiogram results, the clinical isolate was additionally resistant to sulphonamides, trimethoprim, tetracycline, chloramphenicol, gentamicin, tobramycin, netilmicin and spectinomycin and susceptible to ciprofloxacin and imipenem. Rectal swab samples collected from other patients in the same hospital unit concomitantly or during the subsequent 2 weeks failed to grow any ESBL producers.
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The ESBL phenotype was transferred to E. coli at a frequency of 10–5, the transconjugants appearing resistant to broad-spectrum cephalosporins, aminoglycosides, trimethoprim and sulphonamides but susceptible to tetracycline, chloramphenicol and spectinomycin. Plasmid DNA analysis revealed that the transconjugants harboured a c. 80 kb plasmid (pSF-1), as did the original isolate.
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 (200–400 copies/cell) may explain this discrepancy.
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Analysis of the flanking sequences of blaSHV-2 in the 3578 bp insert of plasmid pSF-2 revealed 99% identity with homologous regions of plasmids pBP-60-1-2 from Klebsiella ozaenae containing blaSHV-29 and pPAG-KE from Pseudomonas aeruginosa containing blaSHV-5 (GenBank accession no. AF096930). The three ORFs identified (Figure
) shared 34–64% amino acid identity (data not shown) with proteins from E. coli.10 These results strongly suggest an enterobacterial origin of these ORFs. The promoter found upstream of blaSHV-2 [5'-TTGATT-3' (–35 box) and 5'-AAAAAT-3' (–10 box)] was identical to that described by Podbielski et al.,9 which was shown to be a weak E. coli promoter.3,9 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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This work was supported by a grant from the Ministère de la Recherche Université Paris XI (UPRES, JE 2227), Paris, France.
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Notes |
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* Corresponding author. Tel: +33-1-45-21-20-19; Fax: +33-1-45-21-63-40; E-mail: nicolas.fortineau{at}bct.ap-hop-paris.fr
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References |
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Navia, M. M., Capitano, L., Ruiz, J., Vargas, M., Urassa, H., Schellemberg, D. et al. (1999). Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. Journal of Clinical Microbiology 37, 3113–7.
3 . Nüesch-Inderbinen, M. T., Kayser, F. H. & Hächler, H. (1997). Survey and molecular genetics of SHV ß-lactamases in Enterobacteriaceae in Switzerland: two novel enzymes, SHV-11 and SHV-12. Antimicrobial Agents and Chemotherapy 41, 943–9.[Abstract]
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Received 30 August 2000; returned 18 October 2000; revised 10 November 2000; accepted 29 November 2000
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