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New TEM and SHV -lactamase mutants continue to be described, mostly from klebsiellae (4, 5; information can also be found at the website maintained by G. Jacoby and K. Bush entitled Amino acid sequences for TEM, SHV, and OXA extended-spectrum and inhibitor-resistant -lactamases [http://www.lahey.org/studies/webt.htm]). Many of these variants hydrolyze oxyimino-aminothiazolyl cephalosporins and monobactams and are termed extended-spectrum -lactamases (ESBLs); others are resistant to inhibitors. We report here on four cephalosporin-resistant Klebsiella pneumoniae isolates previously found to give a novel profile after PCR-single-strand conformational polymorphism fingerprinting of their bla_SHV genes (12). This finding implied that the isolates might have a new SHV -lactamase gene variant, and this possibility was investigated. The four organisms were collected during a survey of ESBLs in klebsiellae from 35 European intensive care units (9, 12). They were all from elderly male patients treated at a medical intensive care unit of the University Hospital St. Radboud in Nijmegen, The Netherlands, between March and May 1994. Identification and typing data were published previously (12). The isolates, which were cross-reactive by capsular serotyping, gave very similar genomic profiles after pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA (Fig. 1). On this basis, they were inferred to be representatives of a single strain, designated KXR/PN14.

View larger version (107K): [in this window] [in a new window]   FIG. 1.   PFGE profiles of XbaI-digested DNAs from representative isolates of the KXR/PN14 strain. Lane 1, unrelated ESBL-positive K. pneumoniae strain used as a control; lane 2, molecular weight markers; lanes 3 to 6, isolates 736, 733, 726, and 721, respectively.

MICs were determined on Iso-Sensitest agar (Oxoid, Basingstoke, United Kingdom) with inocula of 10⁴ CFU/spot (9). Plasmids were extracted and electrophoresed by the method of Kado and Liu (7). Conjugation was performed by plate mating of logarithmic-phase cultures, with Escherichia coli K-12 J62-1 (lac mutant, Nal^r) as the recipient (8). Isoelectric focusing was performed on polyacrylamide gels, and -lactamase bands were detected with nitrocefin (8). SHV and TEM -lactamase genes were amplified by PCR: the primers used for bla_SHV were SHVa (5'-TCAGCGAAAAACACCTTG-3'; positions 435 to 452 in the sequence of Mercier and Levesque (10), SHVc (5'-ATGCGTTATATTCGCCTGTG-3'; positions 125 to 144), SHVd (5'-GTTAGCGTTGCCAGTGCTCG-3'; positions 989 to 970), SHVe (5'-CCGTTTCCCAGCGGTCAAGG-3'; positions 614-595), SHVf (5'-TTGTGAATCAGCAAAACGCC-3'; positions 38 to 57), and to SHVg (5'-TAAAGGTGCTCATCATGGGA-3'; positions 329 to 310). The primers used for bla_TEM were TEMa (5'-CCGCTCATGATACAATAACC-3'), TEMb (5'-GTATGGCTTCATTCAGCTCC-3'), TEMc (5'-GGAGCTGAATGAAGCCATAC-3'), TEMd (5'-CTGACAGTTACCAATGCTTA-3'), TEMe (5'-ACGGATGGCATGACAGTAAG-3'), and TEMf (5'-CCCAGTGCTGCAATGATACC-3'). The sequences of the PCR products were determined on an automatic sequencer (ABI 377; Perkin-Elmer, Warrington, United Kingdom) by methods described elsewhere (13).

K. pneumoniae strain 726 was used as a representative for -lactamase purification. Overnight growth from 1 liter of Iso-Sensitest broth was diluted into a 10-fold larger volume of the same medium that had been prewarmed to 37°C. After incubation with continuous shaking for 4 h at 37°C, the harvested cells were frozen and thawed three times to give a crude extract, which was clarified by ultracentrifugation at 100,000 × g and 4°C. All subsequent purification was at 4°C. The supernatant obtained after centrifugation was chromatographed on a carboxymethyl Sephadex C-50 column (Pharmacia, Milton Keynes, United Kingdom) which had been equilibrated in 50 mM malonic acid buffer (pH 5.0). This was eluted with the same buffer containing a 0 to 0.5 M NaCl gradient. Eluent fractions containing the SHV -lactamase were dialyzed against 20 mM Tris HCl (pH 8.5) and loaded onto a 16/10 Q-Sepharose High Performance column (Pharmacia) which had been equilibrated in the same buffer and which, after washing, was eluted with the buffer containing a 0 to 0.5 M NaCl gradient. The partially purified SHV -lactamase thereby obtained was stored at 20°C. Hydrolysis of -lactams was examined by UV spectrophotometric assay in 0.1 M phosphate buffer (pH 7.0) at 37°C at the wavelengths detailed previously (8). Inhibition studies were performed as described previously, with a 10-min reaction period for inhibitor and enzyme before addition of 1 mM benzylpenicillin as the reporter substrate (8).

All four members of the KXR/PN14 strain were resistant to ceftazidime and piperacillin and had decreased susceptibilities to aztreonam, cefuroxime, and ceftriaxone compared with the modal MICs for ESBL-negative isolates from the source survey (Table 1). They remained fully susceptible to imipenem, piperacillin-tazobactam, and cefoxitin; with respect to non--lactam agents, they were resistant to gentamicin but remained susceptible to amikacin and ciprofloxacin (Table 1). -Lactamases with pIs of 5.6 and 7.0 were detected in all four isolates, as were plasmids of 154, 66, 5.4, and 4.6 kb. Ceftazidime resistance was transferred to E. coli K-12 J62-1 from isolate 726, which was taken as a representative strain. The transconjugants were resistant to ceftazidime and gentamicin and had reduced susceptibility to other cephalosporins; they gained the pI 5.6 -lactamase and the 154-kb plasmid but not the pI 7.0 enzyme or the other plasmids.

The nucleotide sequence of the SHV -lactamase gene from isolate 726 was determined, together with the sequence of a short upstream region. The deduced amino acid sequence, numbered according to Ambler et al. (1), had two substitutions compared with the sequence of SHV-1: phenylalanine for isoleucine at position 8 (codon change of ATT to TTT) and serine for arginine at position 43 (codon change of CGC to AGC). In addition, the gene had a silent nucleotide substitution of T (thymine) for C (cytosine) at position 481 compared with the bla_SHV-1 sequence of Mercier and Levesque (10). The novel enzyme was designated SHV-14. The change of Arg43Ser is compatible with the lowering of the pI from 7.6 (for SHV-1) to 7.0. The TEM -lactamase from isolate 726 was deduced to have an amino acid sequence in which arginine 164 was replaced by histidine. This enzyme therefore corresponded to TEM-29, a known ESBL (2).

The SHV-14 -lactamase was partially purified by ion-exchange chromatography. The final preparation was free of TEM-29 enzyme, as confirmed by isoelectric focusing. V_max and K_m values are listed in Table 2. The SHV-14 enzyme had very weak activity against cefotaxime, ceftazidime, and aztreonam, but nevertheless, it was more active than SHV-1 against these oxyimino-aminothiazolyl compounds. The V_max value for aztreonam was over five times above those for cefotaxime and ceftazidime and considerably above that reported for the SHV-1 enzyme. Clavulanate and tazobactam were effective inhibitors, with 50% inhibitory concentrations of 0.032 and 0.4 µM, respectively; the corresponding values for the SHV-1 enzyme are reported to be 0.03 and 0.14 µM, respectively (5).

We conclude that SHV-14 is a novel variant within the SHV family. It had higher V_max rates than SHV-1 for oxyimino-aminothiazolyl cephalosporins, but these rates were still less than 2% of those for penicillin. It was more active against aztreonam, but hydrolysis remained inefficient, owing to a high K_m. Transfer was not achieved; hence, no direct measurement of its contribution to resistance was possible. Much of the isolates' resistance doubtless depended on the TEM-29 enzyme, which conferred cephalosporin resistance when transferred to E. coli K-12 J62-1. The mutations in SHV-14 affect residues (amino acids 8 and 43) that are not generally associated with ESBL activity; anyway, residue 8 is cleaved with the signal peptide. The substitution at position 43 is very near the beginning of conserved box I (residues 46 to 50) (6). Both the amino acid substitutions of SHV-14 are shared by the SHV-7 and OHIO-1 enzymes, as is the silent C-to-T mutation at nucleotide 481. SHV-7 -lactamase (3) is an ESBL but has the further amino acid substitutions Gly238Ser and Glu240Lys, both of which facilitate attack on oxyimino-aminothiazolyl cephalosporins. OHIO-1 has 11 additional amino acid changes besides those at positions 8 and 43 (11) and is not considered an ESBL (5). On the basis of the criterion that a group 2be enzyme should hydrolyze one or more oxyimino-aminothiazolyl compounds at >10% the rate at which benzylpenicillin is hydrolyzed (5), placement in group 2b, not group 2be, is also appropriate for the SHV-14 enzyme.

Nucleotide sequence accession number. The GenBank accession number for bla_SHV-14 is AF226622.