An outbreak of a CTX-M-type β-lactamase-producing Klebsiella pneumoniae: the importance of using cefpodoxime to detect extended-spectrum β-lactamases

Sir, CTX-M-type β-lactamases are similar to other extendedspectrum β-lactamases (ESBLs) in that they hydrolyse broadspectrum cephalosporins and aztreonam. The CTX-M-type β-lactamases differ, however, from other ESBLs in acting primarily as cefotaximases. Although there is an increasing incidence of CTX-M β-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 β-lactams showing synergy with clavulanate. This susceptibility pattern matched that described for CTX-M-type β-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 inpatients 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 β-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 β-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.