Antimicrobial susceptibility testing.

Organisms were tested by broth microdilution using Mueller-Hinton broth (BD Biosciences, Sparks, Md.) as described by NCCLS (43) and by disk diffusion using Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) as described by NCCLS (44). Antimicrobial agent powders were obtained from the following sources: amikacin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, chloramphenicol, gentamicin, piperacillin, trimethoprim-sulfamethoxazole, and tetracycline from Sigma Chemical Co., St. Louis, Mo.; aztreonam from Bristol-Myers Squibb, Princeton, N.J.; ceftazidime and tobramycin from Eli Lilly, Indianapolis, Ind.; cefoxitin from Merck, Rahway, N.J.; cefpodoxime from Pharmacia-Upjohn, Kalamazoo, Mich.; clavulanic acid from Smith-Kline Beecham, King of Prussia, Pa.; and tazobactam from Lederle, Pearl River, N.Y. All antimicrobial agent-containing disks were obtained from Fisher Scientific. E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 (45), E. coli HB101, and E. coli DH5α were used for quality control.

Isoelectric focusing of β-lactamases.

Crude cell lysates were prepared by a previously described freeze-thaw procedure (68). Isoelectric focusing was performed as described by Matthew and Harris (37). Cell extracts were loaded onto commercially prepared polyacrylamide gel plates (pH 3.5 to 9.5; Pharmacia LKB, Piscataway, N.J.) and electrophoresed to equilibrium by using an LKB Multiphor II apparatus (Pharmacia LKB). β-Lactamases were visualized by staining the isoelectric focusing gel with a 0.05% solution of nitrocefin (BD Biosciences). The isoelectric points of SHV-29 (7.2), TEM-1 (5.4), and KPC-1 (6.7) were calculated by comparison to TEM-12 (5.25), TEM-3 (6.3), SHV-2 (7.6), and SHV-4 (7.8).

Examination of porin genes and porin expression.

PCR amplifications were performed in a Thermoline Amplitron 1 thermal cycler using Taq polymerase (Pharmacia) with 30 cycles of amplification (1 min at 94°C, 1 min at 55°C, and 1 min at 72°C). The primers used to amplify porin genes were U681 and L1316 (Table ​(Table1).1). U681 and L1316 anneal to sequences conserved in porin genes located 215 and 850 bp downstream of the ompK36 start codon, respectively (14).

TABLE 1

Primers used in this study

Primer	Sequence
omp37, omp36, omp35
U681	5′-CGG TTA CGG CCA GTG GGA ATA-3′
L1316	5′-GAC GCA GAC CGA AAT CGA ACT-3′
pBR322
1	5′-CAC TAT CGA CTA CGC GAT CA-3′
2	5′-ACG ATA GTC ATG CCC CGC GC-3′
blaKPC-1
3	5′-CTG GAG GAC TAT GAC TTC-3′
4	5′-ATA CCA CCC TGA CAG CCG-3′
5	5′-TGT CAC TGT ATC GCC GTC-3′
6	5′-CGG GTT GGA CTC AAG ACG-3′
7	5′-TGA TGC GGT ATT TTC TCC-3′
8	5′-ACT GAC ACT GGG CTC TGC-3′
9	5′-GAG CTG AAC TCC GCC ATC-3′
10	5′-CTC AGT GCT CTA CAG AAA ACC-3′
11	5′-TAA CCT TCG CCC TCA CAG ATA C-3′
12	5′-TAT TTT TCC GAG ATG GGT GAC-3′
13	5′-AGC AGA ACT AGA CGG CGA TAC AGT GAC ATC-3′

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Outer membrane proteins were isolated by sarcosyl extraction of total membrane preparations as described previously (22). Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.) as described by the manufacturer. The proteins were examined on sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide linear gradient gels. For OmpK37 analysis, electrophoresis of outer membrane proteins (OMPs) was performed on 11% acrylamide–0.2% bisacrylamide–0.1% SDS gels (14). Samples were boiled for 5 min in Laemmli's sample buffer before electrophoresis. Gels were visualized by staining with Coomassie Blue R250.

Western blotting of OmpK37, OmpK36, and OmpK35 was performed as follows. Proteins from SDS-polyacrylamide gel electrophoresis (PAGE) gels (4 to 16% acrylamide–0.2% bisacrylamide–0.1% SDS) were transferred to Immobilon-P filters (Millipore) as described previously (14, 22). Filters were blocked in 1% bovine serum albumin in phosphate-buffered saline (PBS). After washing, the filters were incubated with diluted (1:100) anti-OmpK37, anti-OmpK36, or anti-OmpK35 antibody (14, 22) and then with alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G (Sigma; 1:5,000). The filters were developed as described previously (14, 22). All the incubations were carried out at room temperature for 1 h in 1% bovine serum albumin–0.05% Tween 20–PBS, and after incubations with the antiserum, the filters were washed with 0.05% Tween 20–PBS (22).

Plasmid profile analysis and probing.

Plasmid DNA from K. pneumoniae 1534 was isolated using the method described by Portnoy et al. (52). The DNA preparations were electrophoresed on 0.85% agarose gels in the presence of 0.5× TBE buffer (45 mM Tris-HCl, 45 mM boric acid, and 1.25 mM EDTA, pH 8.3) at a constant voltage of 90 V for 15 h at 4°C. Supercoiled plasmid DNAs of pDK9 (165 kb) and R1 (97.6 kb) and the plasmids in E. coli V517 (56.7, 5.8, 4.09, 3.15, 2.83, and 2.2 kb) were used as size standards.

The DNA was transferred from the agarose gel to a positively charged nylon membrane as described by the manufacturer (Zeta-Probe; Bio-Rad Laboratories, Hercules, Calif.) and fixed by baking for 3 h at 80°C. The DNA on the filter was hybridized with a 1,010-bp digoxigenin-labeled bla_KPC-1 DNA probe. Hybridization (at 65°C for 15 h) was performed using the Genius nonradioactive nucleic acid labeling and detection system (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) according to the manufacturer's protocol. The plasmids pDK9 and the plasmids in E. coli V517 were used as negative controls. Purified K. pneumoniae 1534 DNA was the positive control.

Carbapenem inactivation assay.

In order to determine whether resistance to imipenem and meropenem was likely caused by production of a β-lactamase, a disk diffusion bioassay was performed. A suspension of E. coli DH5α equivalent to a 0.5 McFarland standard was inoculated on a Mueller-Hinton agar plate as for disk diffusion. Then, five imipenem or meropenem disks were applied evenly spaced on the plate, four on the periphery and one in the center of the plate. A suspension of the organism to be tested for the presence of carbapenemase was adjusted to the turbidity of a 0.5 McFarland standard, and a loop was used to make a 15-mm streak on each side of one imipenem or meropenem disk on the periphery of the plate (the center disk served as the control). Four different organism suspensions were used on each plate. The plates were incubated at 37°C for 18 to 20 h. Alterations in shape of the zones of inhibition around the test organism were considered to be indications of carbapenemase activity. Negative controls for carbapenemase production included E. coli HB101 and K. pneumoniae ATCC 13883.

Filter mating.

The filter mating protocol described by McDougal et al. (38) was used, except that incubation was performed at both 30 and 37°C. E. coli HB101 was used as the recipient.

Transformation.

Plasmid DNA prepared from K. pneumoniae 1534 via a Qiagen plasmid midi-prep kit (Qiagen, Chatsworth, Calif.) was electroporated into E. coli HB101 as described previously (60). Transformants were selected on Luria-Bertani agar containing 120 μg of streptomycin/ml and 1.5 μg of imipenem/ml.

Cloning of bla_KPC-1.

Total cellular DNA was isolated from K. pneumoniae 1534 using a Qiagen plasmid midi-prep kit and was partially digested with BamHI and HindIII (Gibco BRL, Gaithersburg, Md.). A derivative of pBR322, pBR322-catI (in which bla_TEM was replaced by catI), was constructed and used as the vector. The partially digested DNA was ligated into the corresponding sites of the vector by using T4 DNA ligase (Gibco BRL) and electroporated into E. coli DH5α. Clones were selected on Luria-Bertani agar plates containing 40 μg of chloramphenicol/ml and 1.5 μg of imipenem/ml. The initial clone, containing a 7.5-kb insert from K. pneumoniae 1534, was reduced in size to 3.4 kb via complete digestion with BamHI and religation into the same site of pBR322-catI by using T4 ligase. The plasmid DNA, pBR322-catI-bla_KPC-1 containing the cloned bla_KPC-1, was prepared with a QIAprep Spin plasmid kit (Qiagen) and used in the DNA sequencing reactions. The cloned fragment was sequenced as described below by using pBR322-derived primers (Table ​(Table1,1, primers 1 and 2). Other primers used to complete the sequencing of the 3.4-kb insert are listed in Table ​Table11 (primers 3 to 12).

bla_SHV-, bla_TEM-, and bla_KPC-1-specific PCR.

The primers and the PCR conditions used for amplification of bla_SHV and bla_TEM were those described by Rasheed et al. (56). The novel β-lactamase gene, bla_KPC-1, was amplified from the parent strain, K. pneumoniae 1534, by using primers 5 and 10 (Table ​(Table1).1). The PCRs (total volume, 100 μl) contained 0.5 μM (each) primers, 250 μM deoxynucleoside triphosphates, 2 mM MgCl₂, and 2.5 U of Taq DNA polymerase prepared in 1× reaction buffer supplied by the manufacturer (Perkin-Elmer, Applied Biosystems Division [PE-ABI], Foster City, Calif.). The reactions were amplified in a GeneAmp PCR System 9600 thermal cycler (PE-ABI). Cycling parameters were 5 min at 95°C, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 30 s, and extension at 72°C for 1 min 30 s. The PCR amplification was ended by a final extension cycle at 72°C for 10 min.

Sequencing.

Sequencing of the PCR products for bla_SHV, bla_TEM, and bla_KPC-1 was performed after purification of the PCR products with a QIAquick PCR purification kit (Qiagen). bla_KPC-1 was initially sequenced from plasmid DNA (pBR322-catI-bla_KPC-1). Cycle sequencing reactions were performed in a GeneAmp PCR System 9600 thermal cycler with an ABI Prism dRhodamine Terminator cycle sequencing ready reaction kit according to instructions provided by the vendor (PE-ABI). Sequencing reaction products were purified on Centri-Sep spin columns (Princeton Separations, Adelphia, N.J.) and analyzed on an ABI Prism 377 DNA sequencer (PE-ABI).

To eliminate errors due to PCR amplification, leading and lagging strands were sequenced from two independent PCR products for all three β-lactamase genes. The sequences obtained from PCR products were also compared to the sequences obtained from two independent clones (both leading and lagging strands) of bla_KPC-1.

DNA sequencing data were analyzed by DNASIS for Windows (Hitachi Software Genetic Systems, San Francisco, Calif.). The DNA and protein sequences of other β-lactamases were from the European Molecular Biology Laboratory and the Swiss-Prot data banks. BLAST and BLASTX programs from the web site of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov /BLAST/) were used to identify bla_KPC-1. The amino acid sequences of known class A β-lactamases (Nmc-A [42], IMI-1 [57], and Sme-1 [41]) were aligned by using the multiple alignment (Higgins-Sharp) option of DNASIS for Windows. The restriction map of the 3.4-kb cloned fragment was determined from the sequencing data by using DNASIS. The dendrogram presented in Fig. ​Fig.66 was generated by DNASIS from the alignment of the amino acid sequence of KPC-1 with known β-lactamases (Higgins-Sharp) representative of class A β-lactamases, including CARB-3 (27), PSE-1 (24), SHV-1 (39), LEN-1 (2), TEM-1 (65), MEN-1 (6), OXY1 (19), CITDI (50), YENT (61), Nmc-A (42), IMI-1 (57), Sme-1 (41), L2 (71), ROB-1 (31), and BRO-1 (D. Beaulieu, L. Piche, T. R. Parr, Jr., K. Roeger-Lawry, P. Rosteck, and P. H. Roy, β-lactamase BRO-1 precursor [penicillinase], gi:24975813, GenBank, 1996); representative of class B β-lactamases, including IMP-1 (48) and CfiA (69); representative of class C β-lactamases (ACT-1 [7]); and representative of class D β-lactamases (OXA-1 [49]).

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FIG. 6

Dendrogram showing similarity of 20 β-lactamases. The dendrogram was constructed by using DNASIS for Window's multiple alignment option (Higgins-Sharp). Sixteen of the β-lactamases are class A enzymes, CARB-3 from P. aeruginosa (27), PSE-1 from P. aeruginosa (24), SHV-1 from E. coli (39), LEN-1 from K. pneumoniae (2), TEM-1 from E. coli (65), MEN-1 from E. coli (6), OXY-1 from Klebsiella oxytoca (19), CITDI from C. diversus (50), YENT from Y. enterocolitica (61), Nmc-A from E. cloacae (42), IMI-1 from E. cloacae (57), Sme-1 from S. marcescens (41), L2 from Stenotrophomonas maltophilia (71), ROB -1 from Haemophilus influenzae (31), and BRO-1 from Moraxella catarrhalis (D. Beaulieu, L. Piche, T. R. Parr, Jr., K. Roeger-Lawry, P. Rosteck, and P. H. Roy, β-lactamase BRO-1 precursor [penicillinase], gi:2497581, Gen Bank, 1996); IMP-1 from S. marcescens (48) and Cfi-A from Bacteroides fragilis (69) were included as representatives of class B (metallo-β-lactamases); ACT-1 from K. pneumoniae (7) represents class C, AmpC β-lactamases; and OXA-1 from E. coli (49) represents class D enzymes.

Transcriptional start site of bla_KPC-1.

The transcriptional start site of bla_KPC-1 was mapped by primer extension. Total RNA was isolated from parent strain K. pneumoniae 1534 and E. coli DH5α harboring the plasmid DNA encoding KPC-1 by using the SV total RNA isolation system (Promega Corporation, Madison, Wis.) as described by the manufacturer. Primer number 13 (Table ​(Table1)1) was labeled at the 5′ end by [γ-³²P]ATP (3,000 Ci/mmol, 10 mCi/ml). The primer labeling and the primer extension reactions were carried out by using primer extension system avian myeloblastosis virus reverse transcriptase (Promega) as described by the manufacturer. The sequencing reactions were performed by using the same primer with Promega's fmol DNA sequencing system as described by the manufacturer. The plasmid DNA encoding KPC-1 was used as template in the sequencing reactions. The primer extension and sequencing reaction products were run on an 8% denaturing polyacrylamide gel containing 7 M urea in 1× TBE (60). The gel was dried and exposed to a PhosphorImager intensifying screen for 5 h and analyzed by using the PhosphorImager system (Molecular Dynamics) with ImageQuant software.

β-lactamase purification.

The cloned KPC-1 β-lactamase was purified for kinetic analysis from E. coli strain HY122 (DH5α/pBR322-catI-bla_KPC-1). Three 1-liter cultures of trypticase soy agar supplemented with 1 μg of imipenem/ml and 40 μg of chloramphenicol/ml were grown overnight at 37°C. Bacteria were harvested by centrifugation and washed with 50 mM phosphate buffer, pH 7.0. The pellets were resuspended in 10 ml of 0.2 M sodium acetate, pH 5.5, and subjected to five freeze-thaw cycles (68). The lysate was centrifuged at 20,000 × g, and the β-lactamase activity of the supernatant was separated by column chromatography through Sephadex G-100 in 50 mM phosphate buffer, pH 7.0. Protein in peak fractions containing nitrocefin-hydrolyzing activity was precipitated with 90% ammonium sulfate; pellets were resuspended and dialyzed in 20 mM Tris, pH 7.8, at 4°C. The KPC-1 β-lactamase was eluted from a Q-Sepharose anion-exchange column in 20 mM Tris, pH 7.8, with a 0 to 0.5 M NaCl gradient. The protein concentration of the Q-Sepharose fractions was determined with the Pierce BCA protein assay. The purity of the KPC-1 preparation was determined by scanning densitometry of a colloidal blue-stained NuPAGE 10% bisacrylamide–Tris gel.

Kinetic studies.

Initial hydrolysis rates were measured in 50 mM phosphate buffer (pH 7.0) on a Shimadzu UV-1601 spectrophotometer at 25°C (68). K_m and V_max values were obtained by averaging results from Lineweaver-Burk, Eadie-Hofstee, Hanes-Woolf, and Cornish-Bowden direct linear plot analyses. Substrates were assayed on at least two separate days, with cephaloridine included as a reference each day. Inhibition of hydrolysis of 100 μM nitrocefin was measured after a 5-min preincubation of enzyme with inhibitor in 100 μl of phosphate buffer (pH 7.0). Fifty percent inhibitory concentrations were determined from inhibition graphs of percent control activity versus concentration of inhibitor.

Imipenem and meropenem resistance involves production of β-lactamase.

Isoelectric focusing of K. pneumoniae 1534 revealed three β-lactamases, with pIs of 7.2, 6.7, and 5.4 (Fig. ​(Fig.1,1, lane 3) (the extra bands between pIs 6.7 and 5.4 in lanes 1 to 3 are presumably degradation products of the pI 6.7 β-lactamase). To determine whether resistance to carbapenems could be attributed to the production of a β-lactamase, a disk diffusion carbapenem inactivation assay was performed. The assay was positive (Fig. ​(Fig.2),2), suggesting that a β-lactamase was involved in hydrolysis of imipenem and meropenem in K. pneumoniae 1534 and in the E. coli HB101 transformant (Fig. ​(Fig.2,2, disks 1 and 5, respectively). This β-lactamase was named K. pneumoniae carbapenemase 1, or KPC-1. The presence of EDTA did not inhibit the activity of the β-lactamase, nor did the addition of ZnCl₂ enhance the β-lactamase activity against imipenem or meropenem (data not shown).

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FIG. 1

Isoelectric focusing patterns of carbapenem-resistant K. pneumoniae 1534. The gel was stained with nitrocefin, which is specific for β-lactamases. Lane 1, cell lysate prepared from the imipenem-resistant E. coli DH5α containing the bla_KPC-1 gene on pBR322-catI; lane 2, cell lysate prepared from imipenem-resistant E. coli HB101 that was transformed with K. pneumoniae 1534 DNA; lane 3, cell lysate prepared from K. pneumoniae 1534; lane 4, cell lysates prepared from strains producing SHV-2 (pI of 7.6), TEM-3 (pI of 6.3), and TEM-1 (pI of 5.4); lane 5, cell lysates prepared from strains producing TEM-1 (pI of 5.4) and SHV-4 (pI of 7.8); lane 6, cell lysates prepared from strains producing TEM-12 (pI of 5.25), TEM-10 (pI of 5.6), SHV-3 (pI of 6.8), and SHV-2 (pI of 7.6). The extra bands between pIs 6.7 and 5.4 are presumably degradation products of KPC-1 (lanes 1 to 3). The pIs of the β-lactamases were calculated by using the known pIs of TEM-12 (pI of 5.25), TEM-1 (pI of 5.4), TEM-10 (pI of 5.6), TEM-3 (pI of 6.3), SHV-3 (pI of 6.8), SHV-2 (pI of 7.6), and SHV-4 (pI of 7.8).

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FIG. 2

(A) Inhibition assay with imipenem (IPM); (B) inhibition assay with meropenem (MEM). Disks: 1, lysate of K. pneumoniae 1534 (parent strain); 2, lysate of E. coli HB101/pBR322-catI; 3, disk control with imipenem (A) or meropenem (B); 4, lysate of K. pneumoniae ATCC 13883; 5, lysate of E. coli HB101/pBR322-catI-bla_KPC-1 clone.

PCR and DNA sequence analysis detected the presence of bla_SHV and bla_TEM.

Isoelectric focusing results suggested the presence of bla_SHV (pI 7.2) and bla_TEM (pI 5.4) derivatives (Fig. ​(Fig.1,1, lane 3). PCR analysis using bla_SHV- and bla_TEM-specific primers confirmed the presence of these genes in K. pneumoniae 1534 (data not shown). DNA sequencing results identified the genes as bla_TEM-1 and a novel bla_SHV-29 (H. Yigit, G. J. Anderson, and F. C. Tenover, unpublished data).

Cloning of the bla_KPC-1 gene from the E. coli DH5α transformant.

The filter mating results showed that the carbapenem resistance determinant in K. pneumoniae 1534 was not encoded by a conjugative plasmid. However, electroporation results demonstrated that the gene encoding resistance to carbapenems, extended-spectrum cephalosporins, and aztreonam was located on an approximately 50-kb plasmid (Fig. ​(Fig.3A,3A, lane 4).

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FIG. 3

(A) Plasmid profiles of K. pneumoniae 1534 and an E. coli transformant on 0.85% agarose gel. Lane 1, plasmid RP4 (57-kb); lane 2, plasmid pDK9 (165-kb); lane 3, total DNA prepared from an E. coli HB101 transformant; lane 4, total DNA isolated from K. pneumoniae 1534; lane 5, plasmid R1 (97-kb). Chr., chromosomal. (B) Southern blot of the gel shown in panel A after hybridization with a 1,010-bp bla_KPC-1-specific probe. Lane 1, RP4 DNA; lane 2, pDK9 DNA; and lane 5, R1 DNA served as negative controls for the probe. DNA isolated from K. pneumoniae 1534 (lane 4) was used as a positive control. Lane 3, DNA isolated from an E. coli HB101 transformant of K. pneumoniae 1534 DNA that was imipenem resistant.

A bla_KPC-1-specific DNA probe confirmed the location of bla_KPC-1 on the 50-kb plasmid (Fig. ​(Fig.3B,3B, lanes 3 and 4). Resistance to chloramphenicol, gentamicin, tobramycin, and trimethoprim-sulfamethoxazole was not linked to imipenem and meropenem resistance (Table ​(Table2,2, see HB101 transformant).

Neither bla_SHV-29 nor bla_TEM-1 was present in the transformants examined by PCR analysis. However, bla_KPC-1-specific products were generated from the E. coli transformants (data not shown), which was consistent with the isoelectric focusing data showing only one β-lactamase with a pI of 6.7 (Fig. ​(Fig.1,1, lane 2).

The antibiogram of the E. coli DH5α bla_KPC-1 clone (which harbors the plasmid pBR322-catI-bla_KPC-1, which contains the 3.4-kb cloned insert from K. pneumoniae 1534) is shown in Table ​Table2.2. The MICs are consistent with those for E. coli HB101 transformants containing the 50-kb plasmid encoding KPC-1 (Table ​(Table2).2). This demonstrates that the β-lactamase gene located on the 3.4-kb fragment is responsible for the resistance to carbapenems, extended-spectrum cephalosporins, and aztreonam. E. coli DH5α(pBR322-catI-bla_KPC-1) encoded a single β-lactamase with a pI of 6.7, as shown by isoelectric focusing (Fig. ​(Fig.1,1, lane 1).

Sequence analysis of bla_KPC-1.

The nucleotide sequence of the carbapenemase gene was determined from pBR322-catI-bla_KPC-1. The nucleotide sequence of bla_KPC-1 (Fig. ​(Fig.4)4) did not show significant similarity to those of any other β-lactamase genes or other sequences in GenBank.

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FIG. 4

Nucleotide and deduced amino acid sequences of the novel class A carbapenemase KPC-1 isolated from K. pneumoniae 1534. The −10 and −35 regions of the putative promoter are underlined. The transcription start site indicated by the mRNA primer extension study is marked as +1. RBS indicates a potential ribosome-binding site (62). The HindIII recognition site and the conserved amino acid residues for class A carbapenemases are underlined (26, 35, 36). The start and stop codons for bla_KPC-1 are marked with asterisks (59).

The location of the bla_KPC-1 open reading frame (ORF) was verified by HindIII subcloning of the 3.4-kb cloned fragment. The fragment contained one HindIII site that cleaves in the predicted ORF of bla_KPC-1 (Fig. ​(Fig.4).4). Both of the HindIII sub-fragments of the original 3.4-kb fragment were subcloned, but neither encoded a functional β-lactamase. Therefore, this region was proven to represent the ORF for bla_KPC-1.

bla_KPC-1 contained an 879-bp coding region which encoded a 32,230-Da protein containing 293 amino acids (Fig. ​(Fig.4).4). The protein contains a serine-serine-phenylalanine-lysine (S-S-F-K) and a lysine-threonine-glycine (K-T-G) motif. These sequences, S-X-X-K and K-T-G, are characteristic of class A serine β-lactamases (25, 26). Other conserved residues among class A carbapenemases (55, 63) are shown in bold and underlined in Fig. ​Fig.44.

A six-nucleotide sequence located eight nucleotides upstream from the ATG initiation codon (AAGGAA) was identified as a possible ribosome-binding site (59, 62) for bla_KPC-1. mRNA primer extension results identified the GATTAC sequence as the −10 region and determined the mRNA starting site for bla_KPC-1 (the mRNA start site is denoted +1 in Fig. ​Fig.4).4). The primer extension results also suggested GATAAT as the putative −35 region (Fig. ​(Fig.44).

Amino acid sequence similarity to other β-lactamases.

KPC-1 had the highest similarity to the class A carbapenem-hydrolyzing β-lactamases, in particular to Sme-1 (45% identical) from Serratia marcescens (41). The amino acid sequences of three known class A carbapenemases were aligned with the amino acid sequence of KPC-1 (Fig. ​(Fig.5).5). The regions critical for catalysis are shown in Fig. ​Fig.55 (25, 26, 35, 36, 55). The residues known or suggested to be important for class A carbapenemase activity by Raquet et al. (55) are marked by asterisks (C69, S70, K73, H105, S130, R164, E166, N170, D179, R220, K234, S237, and C238). Interestingly, H105 and S237 were not conserved in KPC-1. KPC-1 contained a tryptophan at position 105 and a threonine at position 237. KPC-1 also showed similarity to some other class A β-lactamases, including Nmc-A (44%) (42), IMI-1 (43%) (57), OXY-1 (39%) (19), and MEN-1 (34%) (6). These results, in combination with the kinetic data presented in the next section, place KPC-1 in Bush functional group 2f (8), together with other class A carbapenemases.

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FIG. 5

Alignment of the amino acid sequence of KPC-1 with that of Sme-1 from S. marcescens S6 (41), Nmc-A from E. cloacae NOR1 (42), and IMI-1 from E. cloacae (57). Dashes indicate the gaps that were inserted to optimize the alignment. The numbering is from Ambler et al. and Sykes (1, 67). The conserved domains of class A β-lactamases are underlined (12, 26, 35, 36, 55, 63, 66). The residues suggested to play a critical role for carbapenemase activity are marked by asterisks and underlined (63, 66). The positions where the KPC-1 sequence diverges from these conserved residues (positions 105 and 237) are indicated in italics.

A dendrogram was generated (Fig. ​(Fig.6)6) from the amino acid sequence alignment of KPC-1 with other class A β-lactamases. This shows that KPC-1 is more closely related to the subgroup including YENT (61), CITDI (50), OXY-1 (19), and MEN-1 (6) than to TEM-1 (65) and SHV-1 (39). This finding is in agreement with the results presented for Sme-1 by T. Nass et al. (41). KPC-1 had low amino acid sequence similarity to β-lactamases of class B, e.g., IMP-1 (48) and Cfi-A (69); class C, e.g., AmpC and ACT-1 (7); and class D, e.g., OXA-1 (49) (Fig. ​(Fig.6).6).

Kinetic parameters.

The kinetic parameters for KPC-1 are summarized in Table ​Table3.3. The KPC-1 enzyme used in these studies was approximately 90% pure. KPC-1 hydrolyzed β-lactams from the penicillin, cephalosporin, carbapenem, and monobactam groups. The highest k_cat values were obtained with cephaloridine, which demonstrated a k_cat value approximately four times higher than that for cephalothin or nitrocefin and three times higher than that for ampicillin. Piperacillin, benzylpenicillin, and cloxacillin, the other penicillins tested, had similar k_cat values approximately 10 to 14 times lower than that of cephaloridine. KPC-1 showed hydrolytic activity against the carbapenems; hydrolysis of imipenem occurred at a rate approximately 25 times slower than that of cephaloridine. Meropenem had a k_cat value four times lower than that for imipenem. Hydrolysis rates for cefotaxime and ceftazidime were 24 and 3,370 times lower than the value obtained for cephaloridine. Between the two extended-spectrum cephalosporins tested, cefotaxime had the highest k_cat value, approximately 140 times higher than the k_cat value for ceftazidime. Of the 13 substrates used in these experiments, cefoxitin and ceftazidime had the lowest hydrolysis rates.

TABLE 3

Substrate profile for KPC-1

Substrate	kcat (s−1)	Relative kcat	Km (μM)	Relative kcat/Km
Cephaloridine	340±16	100	560±97	100
Cephalothin	75±7	22	53±0.3	240
Nitrocefin	78±2	23	24±5	530
Cefotaxime	14±1	4.2	160±19	15
Cefoxitin	0.26±0.01	0.08	120±17	0.4
Ceftazidime	0.10±0.02	0.03	94±1.8	0.2
Benzylpenicillin	32±1	9.6	23±2.3	230
Ampicillin	110±4	33	130±21	140
Cloxacillin	25±1	7.4	100±35	41
Piperacillin	24±0	7.2	18±3	230
Imipenem	12.4±0	3.7	81±0.4	26
Meropenem	3.0±0.1	0.9	12±2	41.5
Aztreonam	20±1	5.9	310±35	11

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KPC-1 had the highest affinity for meropenem, with a K_m of 12 μM. Other substrates with low K_ms were piperacillin, penicillin, and nitrocefin, with K_ms that ranged from 18 to 24 μM. Cephaloridine had the highest K_m, 560 μM.

Hydrolytic efficiencies, measured by k_cat/K_m, revealed that penicillin was hydrolyzed by KPC-1 approximately two times more efficiently than cephaloridine. Nitrocefin had the highest catalytic efficiency of the substrates tested, with a value five times that of cephaloridine. The hydrolytic efficiencies of KPC-1 for imipenem and meropenem were substantially less than that of cephaloridine (26 and 42%, respectively). Cefotaxime was hydrolyzed the most efficiently of the three extended-spectrum cephalosporins tested, with a k_cat/K_m value of 15% that for cephaloridine. The hydrolytic efficiencies for cefoxitin and ceftazidime were 250 and 500 times lower than that for cephaloridine, respectively.

Clavulanic acid and tazobactam both inhibited the KPC-1 β-lactamase, with 50% inhibitory concentrations of 10.5 ± 1.3 μM for clavulanic acid and 0.374 ± 0.034 μM for tazobactam (data not shown). Tazobactam was a better inhibitor of KPC-1 than was clavulanic acid. No inhibition was observed when the enzyme was tested with 5 mM EDTA at pH 7.0.

H.Y. is a recipient of an American Society for Microbiology-National Center for Infectious Diseases postdoctoral fellowship.

We thank John E. McGowan, Jr., from Rollins School of Public Health of Emory University for providing the strains from project ICARE. We give special thanks to W. Reznikoff for his assistance with the primer extension studies and his suggestions on determination of the promoter region. Also, we thank A. Glasgow for her helpful suggestions on experimental design, J. K. Rasheed for his suggestions on the manuscript, N. Clark for suggestions regarding Southern blots, and J. Swenson for her help in obtaining the required media and antimicrobial agents for this study.