JAC Advance Access originally published online on December 12, 2002
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Journal of Antimicrobial Chemotherapy (2003) 51, 195-196
© 2003 The British Society for Antimicrobial Chemotherapy
Correspondence |
An outbreak of a CTX-M-type ß-lactamase-producing Klebsiella pneumoniae: the importance of using cefpodoxime to detect extended-spectrum ß-lactamases
1 Department of Microbiology, City Hospital, Birmingham B18 7QH; 2 Division of Immunity and Infection, Medical School, University of Birmingham, Birmingham B15 2TT, UK
Keywords: CTX-M-type ß-lactamase, Klebsiella pneumoniae
Sir,
CTX-M-type ß-lactamases are similar to other extended-spectrum ß-lactamases (ESBLs) in that they hydrolyse broad-spectrum cephalosporins and aztreonam. The CTX-M-type ß-lactamases differ, however, from other ESBLs in acting primarily as cefotaximases. Although there is an increasing incidence of CTX-M ß-lactamases worldwide1 they have not yet been reported in the UK. We describe our preliminary findings from a study into a suspected outbreak involving a CTX-M-producing Klebsiella pneumoniae in Birmingham, UK. The study highlights the importance of using the correct cephalosporin to screen for CTX-M-type ESBLs.
In common with other diagnostic laboratories, screening of Escherichia coli and K. pneumoniae for ESBLs at City Hospital was carried out by disc diffusion using ceftazidime (30 µg disc) as the indicator cephalosporin. During June 2001, it was noticed that a few isolates of K. pneumoniae were borderline resistant to ceftazidime by BSAC disc diffusion methodology.2 The MICs of ceftazidime for the isolates were between 0.5 and 2.0 mg/L by agar dilution MIC. These MICs are equal to or lower than the BSAC susceptible breakpoint of 2.0 mg/L, but higher than the mode MIC of susceptible strains. When retested using a double disc diffusion test for detecting ESBL-producing bacteria,3 the isolates were found to be resistant to cefpodoxime and cefotaxime, both ß-lactams showing synergy with clavulanate. This susceptibility pattern matched that described for CTX-M-type ß-lactamase producers.3
Over 8 months (July 2001 to February 2002), we collected 36 K. pneumoniae isolates with the same susceptibility pattern (cefpodoxime and cefotaxime resistant; ceftazidime susceptible) from 33 patients. Twenty-five of the isolates were from urine, the remainder were from assorted samples, including blood and sputum. Most patients were either in-patients or visited outpatient clinics at City Hospital (15 different wards and departments). Two patients had been seen in GP surgeries. The isolates were identified as K. pneumoniae by API20E (BioMérieux, Basingstoke, UK). The susceptibilities of the isolates to cefotaxime, ceftazidime and aztreonam, in the presence or absence of clavulanic acid (4 mg/L), were determined by agar dilution MIC.4 PCR amplification was used to detect the presence of a CTX-M-type ß-lactamase using primers and conditions published previously.5 Direct sequencing of the PCR amplimers was carried out using the same primers (MWG Biotech Ltd, Milton Keynes, UK). Nine randomly selected isolates were characterized by pulsed-field gel electrophoresis (PFGE). For PFGE, total DNA was prepared as described by Gouby et al.6 XbaI-digested DNA was electrophoresed by a CHEF DR-II system (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK) with a pulse time of 10–70 s at 6 V/cm for 24 h cooled to 10°C. Plasmids were extracted from isolates using a commercial kit following the manufacturer’s method (Qiagen Ltd, Crawley, UK) and separated by electrophoresis on a 0.8% agarose gel. Following transfer to a nylon membrane DNA was hybridized with an alkaline phosphatase-labelled probe produced using the PCR amplimers from one of the isolates (H595) and AlkPhos direct labelling kit (Amersham, UK).
The MICs for the 36 isolates are shown in Table 1. Using BSAC breakpoints, all the isolates would be considered resistant to cefotaxime, but susceptible to ceftazidime and aztreonam (Table 1). The MICs of the three compounds were significantly reduced by clavulanic acid. A PCR amplimer of 
600 bp was obtained from all 36 isolates. The nucleotide sequences of the amplimers from two of the isolates, H595 and H610, were found to be identical. A comparison of the nucleotide sequence of the amplimers with known ß-lactamase sequences (SwissProt and EMBL) showed the sequences to have 99% homology with an equally sized intergenic region of blaCTX-M-25 described in E. coli (accession no. AF518567). The PFGE patterns for all nine of the randomly selected isolates were indistinguishable. The plasmids from five of these isolates were examined; all five contained a similarly sized plasmid of 50 kb, which hybridized with the CTX-M probe.
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Although not all the isolates were examined extensively, our preliminary findings suggest that a single strain of K. pneumoniae harbouring a blaCTX-M-like gene encoding plasmid was responsible for the outbreak. This is the first report of an outbreak involving a CTX-M ß-lactamase-producing bacteria in the UK. Although the MIC of ceftazidime for the CTX-M-producing isolates was increased compared with fully susceptible isolates they would still be considered susceptible using BSAC breakpoints. Some of the isolates were also susceptible to ceftazidime by disc diffusion. If ceftazidime susceptibility was used as the sole means of screening for ESBLs, CTX-M-type enzymes could easily be overlooked. A cephalosporin such as cefpodoxime or cefotaxime should be used when screening for CTX-M ESBLs, as suggested by Livermore & Brown.3 It is likely that the incidence of this enzyme will increase. If laboratories do not use appropriate methodologies, CTX-M ESBLs may not be detected, which could have important consequences regarding the treatment of infections caused by CTX-M-producing organisms.
Footnotes
* Corresponding author. Tel: +44-121-507-4228; Fax: +44-121-551-7763; E-mail: n.p.brenwald{at}bham.ac.uk 
References
1 . Navarro, F. & Miro, E. (2002). Update on CTX-M-type ß-lactamases. Reviews in Medical Microbiology 13, 63–73.
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Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.
3 . Livermore, D. M. & Brown, D. F. J. (2001). Detection of ß-lactamase-mediated resistance. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 59–64.[Abstract]
4 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract]
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Gniadkowski, M., Schneider, I., Palucha, A., Jungwirth, R., Mikiewicz, B. & Bauernfeind, A. (1998). Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolysing ß-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrobial Agents and Chemotherapy 42, 827–32.
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Gouby, A., Neuwirth, C., Bourg, G., Bouziges, N., Carles-Nurit, M. J., Despaux, E. et al. (1994). Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a geriatric hospital. Journal of Clinical Microbiology 32, 301–5.
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