1 Laboratoire de Bactériologie, Hôpital Universitaire du Bocage, BP 1542, 21034 Dijon Cedex; 2 Centre Hospitalier, 71018 Mâcon Cedex; 3 CNRS, UBO, MNHN, 29000 Quimper, France
Keywords: CTX-M15, French patient
Sir,
Since the beginning of the 1990s, 26 members of the CTX-M family have been identified.1 These extended-spectrum ß-lactamases (ESBLs) belong to the class A family and most of them have a good hydrolysis for cefotaxime, whereas ceftazidime is a poor substrate. The geographical origin of these enzymes has been much debated recently: South America or Europe?2 For the scientific community, complete characterization of the enzymes is most important (sequence and biochemical properties). The producing strains have been isolated from various areas, but mainly Asia, South America, Africa and Europe.36
In February 2001 a multiresistant strain of Escherichia coli (EC 1) was isolated in a urine sample of a 49-year-old French woman (patient 1) hospitalized from 15 January to 17 February in the traumatology unit of our University Hospital (Dijon, France). The patient was hospitalized following spinal trauma. She had not been abroad for 5 years. The strain had a high level of resistance to all broad-spectrum cephalosporins, and a double-disc synergy test was positive. The pI of the ESBL, determined by analytical isoelectric focusing, was 8.6, an unusual value.
In May 2001, the Mâcon Hospital Laboratory (100 km from Dijon) sent us an ESBL-producing strain of E. coli, isolated from the wound of an open leg fracture of a 36-year-old French woman (patient 2). In January 2001, the patient went to India on honeymoon, where she was in a car accident. She was first hospitalized in an intensive care unit in New Delhi; 2 weeks later she was back in France. After a 10-day stay in the traumatology unit of our hospital (1222 January), she was transferred to Mâcon, where she lives. The main trauma affected one leg, with multiple fractures associated with substance loss. The bacterial cultures of urine, and a biopsy of bone, were negative in Dijon. After the transfer to Mâcon, the doctors noticed a wide surface of tissue necrosis, with absence of wound closure, and clinical signs of infection. An ESBL-producing strain of E. coli (EC 2) was isolated in this wound and sent to us for characterization of the enzyme.
The EC 2 antibiogram was very similar to that of EC 1, and EC 2 also produced an ESBL with pI 8.6. A comparison, by pulsed-field gel electrophoresis, was performed after digestion of total DNA by XbaI, and revealed that both strains had identical profiles. MICs of ß-lactams, determined by a microdilution method, showed that the strains were highly resistant to penicillins (ticarcillin > 2048 mg/L) and broad-spectrum cephalosporins (cefotaxime 1024 mg/L and ceftazidime 64 mg/L). The best inhibition was obtained with clavulanic acid. The strains were also resistant to kanamycin, tobramycin, gentamicin, tetracycline, co-trimoxazole and quinolones. Imipenem and amikacin maintained good activity. Attempts to transfer the ESBL phenotype to E. coli K12-C600 (resistant to sodium azide) or Klebsiella pneumoniae 10031 (resistant to rifampicin) failed.
PCR amplifications were negative for blaTEM and blaSHV, but positive with primers specific for blaCTX-M: P1C (5'-TCGTCTCTTCCAGA-3') and P2C (5'-CAGCTTATTCATCGC-3'), P3C (5'-GCGATGAATAAGCTG-3') and Fin (5'-CCGTTTCCGCTATTA-3'). The entire coding region of the gene was amplified. Both strands of the PCR products were sequenced. Analysis of the sequences revealed that the ESBL produced by the clinical strains EC 1 and EC 2 was identical to CTX-M15.7 This enzyme differs from CTX-M3 by an aspartic acid to glycine substitution at position 240. The same substitution is also encountered in CTX-M16,8 an enzyme that also provides resistance to ceftazidime in the organisms in which it resides. CTX-M15 was first isolated in Japan. The sequence was submitted in November 2000 by Muratami (Genbank accession number AY013478) but not published. In 2001, the same enzyme was characterized by Karim et al.7 in clinical strains of E. coli, and also in Enterobacter aerogenes and K. pneumoniae isolated in New Delhi.
Strains expressing the common CTX-M-type ß-lactamases are easily detected: they are resistant to cefotaxime, whereas the activity of ceftazidime is conserved. In the strains EC 1 and EC 2 all broad-spectrum cephalosporins had high MICs: before the determination of the pI there was no suspicion of CTX-M production.
Further investigations revealed that both patients 1 and 2 had been hospitalized in the same bedroom of a traumatology unit during a single week. Patient 2 was probably colonized by the multiresistant strain of E. coli in an anatomical site where no bacteriological investigations had been performed before she developed a wound infection. This would seem to confirm that she acquired the strain EC 2 during her stay in the New Delhi hospital. The transmission to patient 1 probably occurred during medical procedures. Fortunately, the strain did not spread in our hospital. It is noteworthy that, in the absence of the study of the strain isolated in Mâcon, we would have concluded wrongly that CTX-M15 had emerged in our hospital, whereas it was an imported strain. This finding highlights the necessity of collecting as much information as possible about patients when unusual or new ß-lactamases are isolated.
Footnotes
* Corresponding author. Tel: +33-3-80-29-32-60; Fax: +33-3-80-29-36-67; E-mail: catherine.neuwirth{at}chu-dijon.fr
References
1 . Jacoby, G. & Bush, K. Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant beta-lactamases. [Online.] http://www.lahey.org/studies/webt.htm (23 October 2002, date last accessed).
2
.
Radice, M., Power, P., Di Conza, J., Gutkind, G., Bonnet, R., Sirot, D. et al. (2002). Early dissemination of CTX-M-derived enzymes in South America. Antimicrobial Agents and Chemotherapy 46, 6024.
3
.
Bradford, P. A. (2001). Extended-spectrum ß-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Reviews 14, 93351.
4
.
Kariuki, S., Corkill, J. E., Revathi, G., Musoke, R. & Hart, C. A. (2001). Molecular characterization of a novel plasmid-encoded cefotaximase (CTX-M-12) found in clinical Klebsiella pneumoniae isolates from Kenya. Antimicrobial Agents and Chemotherapy 45, 21413.
5
.
Sabate, M., Tarrato, R., Navarro, F., Miro, E., Verges, C., Barbe, J. et al. (2000). Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing ß-lactamase (CTX-M-9) from Escherichia coli in Spain. Antimicrobial Agents and Chemotherapy 44, 19703.
6
.
Oliver, A., Perez-Dia, J. C., Coque, T. M., Baquero, F. & Canton, R. (2001). Nucleotide sequence and characterization of a novel cefotaxime-hydrolyzing ß-lactamase (CTX-M-10) isolated in Spain. Antimicrobial Agents and Chemotherapy 45, 61620.
7 . Karim, A., Poirel, L., Nagarajan, S. & Nordmann, P. (2001). Plasmid-mediated extended-spectrum ß-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiology Letters 201, 23741.[CrossRef][ISI][Medline]
8
.
Bonnet, R., Dutour, C., Sampaio, J. L. M., Chanal, C., Sirot, D., Labia, R. et al. (2001). Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240Gly. Antimicrobial Agents and Chemotherapy 45, 226975.