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2001.Novel Carbapenem-Hydrolyzing Lactamase, KPC , from a Carbapenem...

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2001.Novel Carbapenem-Hydrolyzing Lactamase, KPC , from a Carbapenem...

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     ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2001, p. 1151?C1161 0066-4804/01/$04.00 0 DOI: 10.1128/AAC.45.4.1151?C1161.2001 Copyright 2001, American Society for Microbiology. All Rights Reserved.

     Vol. 45, No. 4

     Novel Carbapenem-Hydrolyzing -Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

     HESNA YIGIT,1 ANNE MARIE QUEENAN,2 GREGORY J. ANDERSON,1 ANTONIO DOMENECH-SANCHEZ,3 JAMES W. BIDDLE,1 CHRISTINE D. STEWARD,1 SEBASTIAN ALBERTI,4 KAREN BUSH,2 AND FRED C. TENOVER1* Hospital Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 303331; The R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 088692; and Unidad de Investigacion, Hospital Son Dureta, Andrea Doria, Palma de Mallorca, 07014,4 and Area de Microbiologia, Universidad de las Islas Baleares, Crtra. Valldemosa, Palma de Mallorca, 07071,3 Spain

     Received 19 September 2000/Returned for modication 21 November 2000/Accepted 23 January 2001

     A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 g/ml. The -lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three -lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV and blaTEM genes was conrmed by specic PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the -lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel

    carbapenem-hydrolyzing -lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing -lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that puried KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest afnity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present.

    We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing -lactamase, KPC-1, although alterations in porin expression may also play a role.

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     The carbapenems, such as imipenem and meropenem, are used with increasing frequency in the United States and elsewhere for the treatment of multiresistant gram-negative nosocomial pathogens (21, 29, 30). Resistance to carbapenems is uncommon in enteric organisms; however, resistance can arise by three known mechanisms. First, high-level production of a chromosomal AmpC cephalosporinase combined with decreased outer membrane permeability due to loss or alteration of porins can result in carbapenem resistance. This has been shown for Enterobacter cloacae (28, 54), Enterobacter aerogenes (9, 10, 13, 23), Proteus rettgeri (54), Citrobacter freundii (32), Escherichia coli (11, 64), and Klebsiella pneumoniae (5, 7, 16). The second mechanism is production of a -lactamase that is capable of hydrolyzing carbapenems (8, 30, 58) (e.g., IMI-1 [57], IMP-1 [3, 48], Nmc-A [42, 46], Sme-1 [41], and CA [69]). The third mechanism of resistance involves changes in the afnity of the target enzymes, the penicillin binding proteins, for carbapenems (15, 70). In this study, a K. pneumoniae strain manifesting carbapenem resistance was collected through project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) (4, 20)

     and analyzed for its mechanism(s) of carbapenem resistance. The results presented suggest that the carbapenem resistance phenotype of the strain is mainly caused by the production of a novel class A -lactamase, KPC-1.

     MATERIALS AND METHODS Bacterial strains. The carbapenem-resistant strain K. pneumoniae 1534 was collected from a hospital in North Carolina participating in project ICARE (4, 20). Identication of the isolate was conrmed using standard biochemical tests (17). E. coli HB101 [F supE44 lacY1 ara-14 galK2 xyl-5 mtl-1 leuB6 (mcrC-mrr) recA13 rpsL20 thi-1 (gpt-proA)62 hsdSB20 ] (60) was used for electroporation of plasmid DNA isolated from K. pneumoniae 1534 and as a recipient in conjugal mating experiments (38). E. coli DH5 [supE44 lacU169 ( 80 lacZ M15) hsdR17 recA1 gyrA96 thi-1 relA1] was used for cloning the -lactamase and for plasmid DNA preparation of the clone for DNA sequence analysis (60). K. pneumoniae ATCC 13883 (type strain) and K. pneumoniae 37, a carbapenemsusceptible clinical isolate from the Centers for Disease Control and Prevention collection, were used as controls for porin proles. 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

     * Corresponding author. Mailing address: Nosocomial Pathogens Laboratory Branch (G08), Centers for Disease Control and Prevention, 1600 Clifton Rd., Atlanta, GA 30333. Phone: (404) 639-3375. Fax: (404) 639-1381. E-mail: fnt1@cdc.gov. 1151

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     YIGIT ET AL. TABLE 1. Primers used in this study

     Primer Sequence

     ANTIMICROB. AGENTS CHEMOTHER.

     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 xed by baking for 3 h at 80?ãC. The DNA on the lter was hybridized with a 1,010-bp digoxigenin-labeled blaKPC-1 DNA probe. Hybridization (at 65?ãC 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. Puried 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 -lactamase, a disk diffusion bioassay was performed. A suspension of E. coli DH5 equivalent to a 0.5 McFarland standard was inoculated on a Mueller-Hinton agar plate as for disk diffusion. Then, ve 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?ãC 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 lter mating protocol described by McDougal et al. (38) was used, except that incubation was performed at both 30 and 37?ãC. 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 g of streptomycin/ml and 1.5 g of imipenem/ml. Cloning of blaKPC-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 blaTEM 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 DH5 . Clones were selected on Luria-Bertani agar plates containing 40 g of chloramphenicol/ml and 1.5 g 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-blaKPC-1 containing the cloned blaKPC-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 1, primers 1 and 2). Other primers used to complete the sequencing of the 3.4-kb insert are listed in Table 1 (primers 3 to 12). blaSHV-, blaTEM-, and blaKPC-1-specic PCR. The primers and the PCR conditions used for amplication of blaSHV and blaTEM were those described by Rasheed et al. (56). The novel -lactamase gene, blaKPC-1, was amplied from the parent strain, K. pneumoniae 1534, by using primers 5 and 10 (Table 1). The PCRs (total volume, 100 l) contained 0.5 M (each) primers, 250 M 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 amplied in a GeneAmp PCR System 9600 thermal cycler (PE-ABI). Cycling parameters were 5 min at 95?ãC, followed by 35 cycles of denaturation at 95?ãC for 1 min, annealing at 58?ãC for 30 s, and extension at 72?ãC for 1 min 30 s. The PCR amplication was ended by a nal extension cycle at 72?ãC for 10 min. Sequencing. Sequencing of the PCR products for blaSHV, blaTEM, and blaKPC-1 was performed after purication of the PCR products with a QIAquick PCR purication kit (Qiagen). blaKPC-1 was initially sequenced from plasmid DNA (pBR322-catI-blaKPC-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 puried 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 amplication, leading and lagging strands were sequenced from two independent PCR products for all three -lactamase

     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 4 ????5 5 ????5 6 ????5 7 ????5 8 ????5 9 ????5 10 ????5 11 ????5 12 ????5 13 ????5 -CTG GAG GAC -ATA CCA CCC -TGT CAC TGT -CGG GTT GGA -TGA TGC GGT -ACT GAC ACT -GAG CTG AAC -CTC AGT GCT -TAA CCT TCG -TAT TTT TCC -AGC AGA ACT GAC ATC-3 TAT TGA ATC CTC ATT GGG TCC CTA CCC GAG AGA GAC CAG GCC AAG TTC CTC GCC CAG TCA ATG CGG TTC-3 CCG-3 GTC-3 ACG-3 TCC-3 TGC-3 ATC-3 AAA ACC-3 CAG ATA C-3 GGT GAC-3 CGA TAC AGT

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     Fisher Scientic. E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853 (45), E. coli HB101, and E. coli DH5 were used for quality control. Isoelectric focusing of -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). -Lactamases were visualized by staining the isoelectric focusing gel with a 0.05% solution of nitrocen (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 amplications were performed in a Thermoline Amplitron 1 thermal cycler using Taq polymerase (Pharmacia) with 30 cycles of amplication (1 min at 94?ãC, 1 min at 55?ãC, and 1 min at 72?ãC). The primers used to amplify porin genes were U681 and L1316 (Table 1). U681 and L1316 anneal to sequences conserved in porin genes located 215 and 850 bp downstream of the ompK36 start codon, respectively (14). 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)?C8 to 15% polyacrylamide linear gradient gels. For OmpK37 analysis, electrophoresis of outer membrane proteins (OMPs) was performed on 11% acrylamide?C0.2% bisacrylamide?C0.1% 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?C0.2% bisacrylamide?C0.1% SDS) were transferred to Immobilon-P lters (Millipore) as described previously (14, 22). Filters were blocked in 1% bovine serum albumin in phosphate-buffered saline (PBS). After washing, the lters 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 lters were developed as described previously (14, 22). All the incubations were carried out at room temperature for 1 h in 1% bovine serum albumin?C0.05% Tween 20?CPBS, and after incubations with the antiserum, the lters were washed with 0.05% Tween 20?CPBS (22). Plasmid prole 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?ãC. Supercoiled plasmid DNAs of

     VOL. 45, 2001

     genes. The sequences obtained from PCR products were also compared to the sequences obtained from two independent clones (both leading and lagging strands) of blaKPC-1. DNA sequencing data were analyzed by DNASIS for Windows (Hitachi Software Genetic Systems, San Francisco, Calif.). The DNA and protein sequences of other -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 blaKPC-1. The amino acid sequences of known class A -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. 6 was generated by DNASIS from the alignment of the amino acid sequence of KPC-1 with known -lactamases (Higgins-Sharp) representative of class A -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, -lactamase BRO-1 precursor [penicillinase], gi:24975813, GenBank, 1996); representative of class B -lactamases, including IMP-1 (48) and CA (69); representative of class C -lactamases (ACT-1 [7]); and

    representative of class D -lactamases (OXA-1 [49]). Transcriptional start site of blaKPC-1. The transcriptional start site of blaKPC-1 was mapped by primer extension. Total RNA was isolated from parent strain K. pneumoniae 1534 and E. coli DH5 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 1) was labeled at the 5 end by [ -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. -lactamase purication. The cloned KPC-1 -lactamase was puried for kinetic analysis from E. coli strain HY122 (DH5 /pBR322-catI-blaKPC-1). Three 1-liter cultures of trypticase soy agar supplemented with 1 g of imipenem/ml and 40 g of chloramphenicol/ml were grown overnight at 37?ãC. 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 ve freeze-thaw cycles (68). The lysate was centrifuged at 20,000 g, and the -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 nitrocen-hydrolyzing activity was precipitated with 90% ammonium sulfate; pellets were resuspended and dialyzed in 20 mM Tris, pH 7.8, at 4?ãC. The KPC-1 -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?CTris gel. Kinetic studies. Initial hydrolysis rates were measured in 50 mM phosphate buffer (pH 7.0) on a Shimadzu UV-1601 spectrophotometer at 25?ãC (68). Km and Vmax values were obtained by averaging results from Lineweaver-Burk, EadieHofstee, 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 M nitrocen was measured after a 5-min preincubation of enzyme with inhibitor in 100 l of phosphate buffer (pH 7.0). Fifty

    percent inhibitory concentrations were determined from inhibition graphs of percent control activity versus concentration of inhibitor. Nucleotide sequence accession numbers. The nucleotide sequence of blaKPC-1 reported in this study will appear under GenBank accession number AF297554. The nucleotide sequence of blaSHV-29 reported here will appear under GenBank accession number AF301532.

     KPC-1 CARBAPENEM-HYDROLYZING

     -LACTAMASE

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     RESULTS Antimicrobial susceptibility patterns of K. pneumoniae 1534. The MICs of a variety of antimicrobial agents tested against K.

     pneumoniae 1534 are shown in Table 2. The isolate was resistant to imipenem and meropenem which had MICs of 16 g/ml. The isolate was also resistant to extended-spectrum cephalosporins and aztreonam. Although the MIC of amoxicillin did not decrease when it was tested in combination with clavulanic acid, the MIC of imipenem was reduced from 16 to 2 g/ml when tested in the presence of clavulanic acid (4 g/ml). Similarly, the MIC of meropenem was reduced from 16 to 1 g/ml in the presence of clavulanic acid (Table 2). Imipenem and meropenem resistance involves production of -lactamase. Isoelectric focusing of K. pneumoniae 1534 revealed three -lactamases, with pIs of 7.2, 6.7, and 5.4 (Fig. 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 -lactamase). To determine whether resistance to carbapenems could be attributed to the production of a -lactamase, a disk diffusion carbapenem inactivation assay was performed. The assay was positive (Fig. 2), suggesting that a -lactamase was involved in hydrolysis of imipenem and meropenem in K. pneumoniae 1534 and in the E. coli HB101 transformant (Fig. 2, disks 1 and 5, respectively). This -lactamase was named K. pneumoniae carbapenemase 1, or KPC-1. The presence of EDTA did not inhibit the activity of the -lactamase, nor did the addition of ZnCl2 enhance the -lactamase activity against imipenem or meropenem (data not shown). PCR and DNA sequence analysis detected the presence of blaSHV and blaTEM. Isoelectric focusing results suggested the presence of blaSHV (pI 7.2) and blaTEM (pI 5.4) derivatives (Fig. 1, lane 3). PCR analysis using blaSHV- and blaTEM-specic primers conrmed the presence of these genes in K. pneumoniae 1534 (data not shown). DNA sequencing results identied the genes as blaTEM-1 and a novel blaSHV-29 (H. Yigit, G. J. Anderson, and F. C. Tenover, unpublished data). Cloning of the blaKPC-1 gene from the E. coli DH5 transformant. The lter 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. 3A, lane 4). A blaKPC-1-specic DNA probe conrmed the location of blaKPC-1 on the 50-kb plasmid (Fig. 3B, lanes 3 and 4). Resistance to chloramphenicol, gentamicin, tobramycin, and trimethoprim-sulfamethoxazole was not linked to imipenem and meropenem resistance (Table 2, see HB101 transformant). Neither blaSHV-29 nor blaTEM-1 was present in the transformants examined by PCR analysis. However, blaKPC-1-specic products were generated from the E. coli transformants (data not shown), which was consistent with the isoelectric focusing data showing only one -lactamase with a pI of 6.7 (Fig. 1, lane 2). The antibiogram of the E. coli DH5 blaKPC-1 clone (which harbors the plasmid pBR322-catI-blaKPC-1, which contains the 3.4-kb cloned insert from K. pneumoniae 1534) is shown in Table 2. The MICs are consistent with those for E. coli HB101 transformants containing the 50-kb plasmid encoding KPC-1 (Table 2). This demonstrates that the -lactamase gene located on the 3.4-kb fragment is responsible for the resistance to carbapenems, extended-spectrum cephalosporins, and aztreonam. E. coli DH5 (pBR322-catI-blaKPC-1) encoded a sin-

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     YIGIT ET AL.

     ANTIMICROB. AGENTS CHEMOTHER.

     TABLE 2. Antimicrobial susceptibility patterns of K. pneumoniae 1534, E. coli DH5 clone, and E. coli HB101 transformant

     MIC ( g/ml) Antimicrobial agent(s) K. pneumoniae 1534 (parent) E. coli DH5 E. coli DH5 (pBR322-catI-blaKPC-1) E. coli HB101 transformant containing blaKPC-1 E. coli HB101

     Imipenem Imipenem-clavulanic acida Meropenem Meropenem-clavulanic acid Ampicillin Amoxicillin-clavulanic acid Piperacillin-tazobactam Ceftazidime Cefoxitin Cefpodoxime Cefotaxime Ceftriaxone Aztreonam Gentamicin Tobramycin Trimethoprim-sulfamethoxazole Chloramphenicol

     a

     16 2 16 1 64 32/16 128/4 32 32 16 64 64 64 16 16 8 32

     0.25 0.25 0.25 0.25 2 2/1 1/4 2 2 0.5 1 1 1 0.25 0.25 0.12 4

     8 0.5 4 0.25 64 32/16 128/4 8 16 16 16 32 64 0.25 0.25 0.12 32

     8 0.5 4 0.25 64 32/16 128/4 8 16 16 8 32 64 0.25 0.25 0.12 4

     0.25 0.25 0.25 0.25 4 2/1 1/4 2 4 0.25 1 1 1 0.25 0.25 0.12 4

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     Clavulanic acid was tested at a xed concentration of 4 g/ml.

     gle -lactamase with a pI of 6.7, as shown by isoelectric focusing (Fig. 1, lane 1). Sequence analysis of blaKPC-1. The nucleotide sequence of the carbapenemase gene was determined from pBR322-catIblaKPC-1. The nucleotide sequence of blaKPC-1 (Fig. 4) did not show signicant similarity to those of any other -lactamase genes or other sequences

    in GenBank. The location of the blaKPC-1 open reading frame (ORF) was

     veried by HindIII subcloning of the 3.4-kb cloned fragment. The fragment contained one HindIII site that cleaves in the predicted ORF of blaKPC-1 (Fig. 4). Both of the HindIII subfragments of the original 3.4-kb fragment were subcloned, but neither encoded a functional -lactamase. Therefore, this region was proven to represent the ORF for blaKPC-1. blaKPC-1 contained an 879-bp coding region which encoded a 32,230-Da protein containing 293 amino acids (Fig. 4). The

     FIG. 1. Isoelectric focusing patterns of carbapenem-resistant K. pneumoniae 1534. The gel was stained with nitrocen, which is specic for -lactamases. Lane 1, cell lysate prepared from the

    imipenem-resistant E. coli DH5 containing the blaKPC-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 -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-blaKPC-1 clone.

     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 -lactamases (25, 26). Other conserved residues among class A carbapenemases (55, 63) are shown in bold and underlined in Fig. 4. A six-nucleotide sequence located eight nucleotides upstream from the ATG initiation codon (AAGGAA) was identied as a possible ribosome-binding site (59, 62) for blaKPC-1. mRNA primer extension results identied the GATTAC sequence as the 10 region and determined the mRNA starting site for blaKPC-1 (the mRNA

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