Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wachino, J.-i. Articles by Arakawa, Y. Search for Related Content PubMed PubMed Citation Articles by Wachino, J.-i. Articles by Arakawa, Y.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, February 2006, p. 534-541, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.534-541.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Horizontal Transfer of bla_CMY-Bearing Plasmids among Clinical Escherichia coli and Klebsiella pneumoniae Isolates and Emergence of Cefepime-Hydrolyzing CMY-19

Jun-ichi Wachino,^1,2 Hiroshi Kurokawa,¹ Satowa Suzuki,¹ Kunikazu Yamane,¹ Naohiro Shibata,¹ Kouji Kimura,¹ Yasuyoshi Ike,² and Yoshichika Arakawa^1*

Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo,¹ Department of Bacteriology and Bacterial Infection Control, Gunma University Graduate School of Medicine, Gunma, Japan²

Received 30 June 2005/ Returned for modification 17 August 2005/ Accepted 4 November 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nine Escherichia coli and 5 Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital between 1995 and 1997. All nine E. coli isolates and one K. pneumoniae isolate carried bla_CMY-9, while the other four K. pneumoniae isolates harbored a variant of bla_CMY-9, namely, bla_CMY-19. The pulsed-field gel electrophoresis patterns of the nine CMY-9-producing E. coli isolates were almost identical, suggesting their clonal relatedness, while those of the five K. pneumoniae isolates were divergent. Plasmid profiles, Southern hybridization, and conjugation assays revealed that the genes for the CMY-9 and the CMY-19 ß-lactamases were located on very similar conjugative plasmids in E. coli and K. pneumoniae. The genetic environment of bla_CMY-19 was identical to that of bla_CMY-9. A single amino acid substitution, I292S, adjacent to the H-10 helix region was observed between CMY-9 and CMY-19. This substitution was suggested to be responsible for the expansion of the hydrolyzing activity against several broad-spectrum cephalosporins, and this finding was consistent with the kinetic parameters determined with purified enzymes. These findings suggest that the bla_CMY-19 genes found in the four K. pneumoniae isolates might have originated from bla_CMY-9 gene following a point mutation and dispersed among genetically different K. pneumoniae isolates via a large transferable plasmid.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Resistance to ß-lactam antibiotics in gram-negative bacilli is mainly mediated by the production of ß-lactamases, which are divided into four major molecular classes, classes A, B, C, and D (1, 10). Genes for AmpC (class C) ß-lactamases are generally encoded on the chromosomes in many gram-negative microbes, including Enterobacter spp., Citrobacter freundii, Serratia marcescens, Morganella morganii, and Pseudomonas aeruginosa (27). Chromosomal AmpC enzymes are usually inducible and are often responsible for resistance to cephalosporins (27) as well as to penicillins. Plasmid-mediated class C ß-lactamases have mainly been described in Klebsiella spp., Escherichia coli, and Salmonella spp. throughout the world (25). A cephamycin-resistant Klebsiella pneumoniae strain producing a plasmid-mediated class C ß-lactamase, CMY-1, was first reported in 1989 in Korea (7, 8). Plasmid-mediated class C enzymes are currently divided into at least five clusters (25) on the basis of amino acid sequence similarities, together with their putative progenitor chromosomal AmpC enzymes. In Japan, MOX-1 (16), CMY-8 (unpublished data), CMY-9 (12), CMY-2 (unpublished data), CFE-1 (23), and DHA-1 (unpublished data) have so far been found as plasmid-mediated AmpC ß-lactamases, mainly in nosocomial isolates of the family Enterobacteriaceae.

Between 1995 and 1997, eight additional E. coli isolates and five K. pneumoniae isolates resistant to both oximino-cephalosporins and cephamycins were isolated in the same hospital where the first CMY-9-producing E. coli strain (strain HKYM68) was isolated in 1995 (12). In the present study, the molecular and biochemical mechanisms underlying the multiple-cephalosporin resistance among these 14 isolates as well as their genetic relatedness were elucidated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. Nine E. coli isolates and five K. pneumoniae isolates displaying a high level of resistance to cephalosporins and cephamycins were isolated between 1995 and 1997 in a general hospital in Yamaguchi Prefecture, Japan, and stored in our laboratory. Among these isolates, E. coli strain HKYM68 was previously found to produce CMY-9 (12). Phenotypic identification of each isolate was performed by using a commercial identification system (API 20E system; bioMerieux, Marcy 1'Etoile, France), according to the instructions of the manufacturer.

Phenotypic test for ß-lactamase types. A simple initial screening test for the presumptive identification of the ß-lactamase types in clinical isolates was performed by use of the double-disk synergy test with Kirby-Bauer disks. Two disks which contained ceftazidime (30 µg per disk) or cefotaxime (30 µg per disk) were used in combination with three different disks containing either amoxicillin-clavulanate (20 µg per disk/10 µg per disk), sodium mercaptoacetic acid (3 mg per disk), or 3-aminophenyl boronic acid (APB) (300 µg per disk), which are specific inhibitors of class A, class B, and class C ß-lactamases, respectively (2, 32).

Identification of ß-lactamase genes by PCR and sequencing analyses. The samples were screened by PCR with 12 sets of primers for the detection of TEM- and SHV-derived extended-spectrum ß-lactamases; GES-type, CTX-M-2-type, CTX-M-3-type, and CTX-M-9-type class A ß-lactamases; CMY-1-, CMY-2-, and DHA-1-type class C ß-lactamases; and IMP-1-, IMP-2-, and VIM-2-type class B ß-lactamases. The sets of PCR primers and the amplification conditions used to detect various plasmid-mediated ß-lactamase genes found thus far in Japan have been reported previously (28, 31). The PCR amplicons were electrophoresed on a 2% agarose gel and purified with a MinElute gel extraction kit (QIAGEN K. K., Tokyo, Japan), and both strands were sequenced.

Transfer of ß-lactam resistance. A conjugation experiment was performed by the broth mating method with E. coli strain CSH-2 (metB F^– Rif^r Nal^r) as the recipient. The donor-to-recipient ratio was 1:4, and the mating time was 3 h. Transconjugants were selected on Luria-Bertani (LB) agar plates supplemented with both rifampin (100 µg/ml) and nalidixic acid (50 µg/ml), together with cefotaxime (10 µg/ml) or ceftazidime (10 µg/ml).

Antibiotic susceptibility tests. Susceptibilities to antibiotics were tested by the agar dilution method according to the procedure recommended by the CLSI (formerly the National Committee for Clinical Laboratory Standards) document M7-A5 (24). E. coli ATCC 25922 was used as the control strain for the antimicrobial susceptibility testing.

Isoelectric focusing of ß-lactamases. Bacterial cells were grown in 10 ml of LB broth supplemented with cephalothin (50 µg/ml) and were harvested by centrifugation (4,000 x g for 15 min) The cell pellet was resuspended in 1 ml of 50 mM sodium phosphate buffer. The pI of ß-lactamase was determined as described previously (31).

Pulsed-field gel electrophoresis (PFGE). Total DNA preparations containing both chromosomal and plasmid DNAs were extracted from each isolate and digested overnight with XbaI (New England Biolabs, Beverly, MA) in agarose gel plugs. The digested DNAs were subjected to electrophoresis with a CHEF-DRII drive module (Bio-Rad Laboratory, Hercules, CA), with pulses ranging from 12.5 to 40 s at 6 V/cm for 24 h at 16°C.

Plasmid analysis and Southern hybridization. Large plasmids mediating bla_CMY genes were prepared from clinical isolates and their transconjugants according to the procedure described by Kado and Liu (17) and electrophoresed on a 0.8% agarose gel. The plasmid DNAs of the transconjugants were also prepared by using a QIAGEN midi-prep kit (QIAGEN K. K.), digested with SacI, and then transferred to a nylon membrane (Bio-Rad Laboratories). The 999-bp digoxigenin (DIG)-labeled DNA probes were prepared by using a PCR DIG Probe Synthesis kit (Roche Diagnostics, Tokyo, Japan); and the DNA template was prepared from a bla_MOX-positive E. coli strain HKYM68 (12), together with two PCR primers, primers MOX-F (5'-AAC AAC GAC AAT CCA TCC-3') and MOX-R (5'-TGT TGA AGA GCA CCT GGC-3').

PCR and sequencing analyses of flanking regions of bla_CMY. To determine the genetic environments of the bla_CMY genes, standard PCR amplification experiments and sequencing analyses were performed with an Expand High-Fidelity PCR system (Roche) and several sets of primers, which were designed on the basis of the nucleotide sequences deposited in the EMBL/GenBank/DDBJ databases under accession number AB061794. The resultant PCR products were purified by using a MinElute gel extraction kit (QIAGEN) and were subsequently sequenced with the appropriate primers.

Cloning of bla_CMY-9 and bla_CMY-19 for purification of enzymes. To amplify bla_CMY-9 and bla_CMY-19, conjugative plasmids pK209 and pK466 were used as the template DNA, respectively. A highly reliable PCR amplification was performed with primers CMY-S1 (5'-CAG GGC GTG AGG ATA AAG-3') and CMY-S2 (5'-GGG ACG AGA TAG AGA AAT-3') by using the Expand High-Fidelity PCR system (Roche). Each amplicon was ligated to the pGEM-T vector (Promega, Madison, WI) and subjected to confirmatory sequencing. Selected plasmids with no amplification error, pGEM-CMY-9 and pGEM-CMY-19, which carry bla_CMY-9 and bla_CMY-19, respectively, were digested with XhoI and EcoRI. The resultant fragments were ligated to pBCSK+ (Stratagene, La Jolla, Calif.) restricted with the same enzymes; and competent cells of E. coli strain DH5 [supE44 lacU169 (80 lacZM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 acrAB⁺], purchased from TOYOBO, Co., Ltd, Tokyo, Japan, were transformed by electroporation with the mixture of the constructed plasmids.

Purification of CMY-9 and CMY-19 ß-lactamases. E. coli strain DH5, which harbored pBC-CMY-9 carrying the bla_CMY-9 gene or pBC-CMY-19 carrying the bla_CMY-19 gene, was separately cultured overnight in 2 liters of LB broth containing cephalothin (50 µg/ml) and chloramphenicol (30 µg/ml). The cells were harvested by centrifugation and washed in 50 mM sodium phosphate buffer (pH 7.0). The pellets were resuspended with 10 ml of 20 mM Tris-HCl buffer (pH 7.5) and destroyed with a French press. After low-speed centrifugation (3,300 x g for 15 min) to remove the cellular debris and unbroken cells, the supernatant was again centrifuged at 100,000 x g for 1 h at 4°C. The supernatant containing ß-lactamase was chromatographed through a HiTrap Q HP column (Amersham Biosciences) that had been preequilibrated with 20 mM Tris-HCl buffer (pH 7.5). ß-Lactamase activity was detected in the flowthrough fraction, which was then dialyzed against 50 mM sodium phosphate buffer (pH 6.0). This partially purified fraction was again applied to a HiTrap SP HP column (Amersham Biosciences) that had been preequilibrated with 50 mM sodium phosphate buffer (pH 6.0). The enzymes were eluted with a linear gradient of NaCl in the same buffer. Fractions with ß-lactamase activity were dialyzed against 50 mM sodium phosphate buffer (pH 7.0) and condensed by use of an Ultrafree-15 centrifuge filter device (Millipore Corporation, Bedford, MA). The production of CMY-19 was not enough in the E. coli transformant, so the following method was used. The bla_CMY-19 gene was amplified with primers CMY-F2 (5'-CAT ATG CAA CAA CGA CAA TCC ATC C-3'), which has an NdeI linker (underlined), and CMY-R2 (5'-GAA TTC TCA ACC GGC CAA CTG CGC CA-3'), which has an EcoRI linker (underlined), and the Expand High-Fidelity PCR system (Roche). The amplicon was ligated with a pGEM-T vector (Promega), subjected to confirmatory sequencing, and then excised by digestion with NdeI and EcoRI and subcloned into the expression vector pET29a(+) (Novagen, Madison, WI), which was cleaved with the same enzymes. The constructed expression vector, named pET-CMY-19, was introduced into E. coli BL21(DE3)pLysS [F^– ompT hsdSB (r_B^–m_B^–) gal dcm (DE3) pLysS], which was obtained from Novagen through TAKARA BIO Inc., Kyoto, Japan. The transformant was cultured in 1 liter of LB broth containing kanamycin (50 µg/ml) and chloramphenicol (30 mg/ml) at 37°C. Isopropyl-ß-D-thiogalactopyranoside was added when the culture reached an optical density at 600 nm of 0.55, and the culture was incubated for an additional 6 h at 25°C. CMY-19 was purified by the same methods used for the purification of CMY-9. The purity of the ß-lactamases was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue (CBB) staining. The purified CMY-9 and CMY-19 ß-lactamases were also subjected to isoelectric focusing analysis with an Ampholine PAG plate (Amersham Biosciences) and stained with CBB.

Assay of kinetic parameters. The kinetic parameters of CMY-9 and CMY-19 against various ß-lactam substrates were assayed at 30°C in 50 mM sodium phosphate buffer (pH 7.0) by using an autospectrophotometer (V-550; Nihon Bunko Ltd., Tokyo, Japan). The absorption maxima of the substrates used were as follows: ampicillin, 235 nm; piperacillin, 232 nm; cephalothin, 262 nm; cephaloridine, 297 nm; ceftizoxime, 257 nm; ceftazidime, 274 nm; cefotaxime, 264 nm; cefpirome, 267 nm; cefepime, 275 nm; cefoxitin, 270 nm; cefmetazole, 259 nm; moxalactam, 274 nm; imipenem, 298 nm. K_m and k_cat values were obtained by a Michaelis-Menten plot of the initial steady-state velocities at different substrate concentrations. K_i was determined by the procedure described in our previous study (13), with cephalothin used as a reporter substrate.

Nucleotide sequence accession number. The open reading frame of bla_CMY-19 was deposited in the EMBL/GenBank databases through DDBJ and assigned accession number AB194410.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Properties of nine E. coli and five K. pneumoniae clinical isolates. The MICs of six ß-lactams for the 14 clinical isolates are shown in Table 1. The K. pneumoniae and E. coli clinical isolates exhibited resistance to oximino-cephalosporins and cephamycins but were susceptible to carbapenems, although E. coli HKYM68 also showed resistance to imipenem. In a double-disk synergy test, no synergistic effect of clavulanic acid on the activities of ceftazidime and cefotaxime was detectable in any of the 14 isolates. A lack of metallo-ß-lactamase production was also suggested by the results of the sodium mercaptoacetic acid disk tests. An apparent expansion of the growth inhibitory zone was observed with the 14 clinical isolates only between a disk containing 300 µg of 3-aminophenyl-boronic acid and a disk containing ceftazidime or cefotaxime, suggesting the production of a class C ß-lactamase. These findings indicate that the property of resistance to oxyimino-cephalosporins and cephamycins was likely due to the production of a class C ß-lactamase.

View this table: [in this window] [in a new window]

TABLE 1. MICs for parent strains and their transconjugants

PCR detection of various ß-lactamase genes and sequencing revealed that a K. pneumoniae isolate (HKY209) carried bla_CMY-9, while the other four K. pneumoniae isolates carried bla_CMY-19, a variant gene of bla_CMY-9 (Table 1). A single nucleotide mutation at position 944 was found between bla_CMY-9 and the newly identified bla_CMY-19 gene, and this point mutation resulted in the I292S substitution near the H-10 helix domain in CMY-19, as shown in Fig. 1. All nine E. coli clinical isolates carried both the bla_CMY-9 and the bla_TEM genes (Table 1). The mechanism of imipenem resistance of HKYM68 was not characterized in this work.

View larger version (42K): [in this window] [in a new window]

FIG. 1. Alignments of amino acid residues near the H-10 helix. A partial amino acid sequence alignment of CMY-9 (12), CMY-19 (this study), CMY-11 (21), FOX-1 (15), AmpC of E. cloacae Ear1 and Ear2 (5), AmpC of E. cloacae P99 and HD (6), AmpC of E. coli K-12 and HKY28 (13), and AmpC of S. marcescens S3 and HD (22) is shown. Square boxes show the amino acid substitutions or deletions that are predicted to affect the hydrolyzing activity of cefepime. The conserved motif KTG is underlined. Dashes indicate deletions of amino acid residues. CMY-11- and FOX-type enzymes have a serine residue at amino acid position 292, but no observation about their property against cefepime was described in the articles. The numbering of the amino acid residues is in reference to that of the mature CMY-1 reported by Bauernfeind et al. (7).

Transferability of ß-lactam resistance. The oximino-cephalosporin and cephamycin resistance trait of the five K. pneumoniae was transferred to a recipient E. coli strain (strain CSH-2) at a frequency of 10^–4 to 10^–5 cells per recipient cell by broth mating. Conjugal transfer of the resistance trait from the nine E. coli isolates was also observed at a frequency of about 10^–3 to 10^–4 cells per recipient cell.

PCR analyses confirmed the presence of bla_CMY-9 or bla_CMY-19 in each transconjugant, indicating that these genes are located on transferable plasmids. PCR analysis of the transconjugants also revealed no cotransmission of the bla_TEM gene to the E. coli transconjugants that harbored the bla_CMY-9 gene.

PFGE analysis. The PFGE patterns of the five K. pneumoniae isolates after XbaI digestion were highly variable (Fig. 2A), which revealed their clonal diversity. In contrast, the PFGE patterns of the E. coli isolates were very similar to one another (Fig. 2B), which revealed their clonal relatedness.

View larger version (107K): [in this window] [in a new window]

FIG. 2. PFGE analysis of K. pneumoniae and E. coli isolates. (A) Lanes: M, PFGE marker; 1, K. pneumoniae HKY209; 2, K. pneumoniae HKY327; 3, K. pneumoniae HKY363; 4, K. pneumoniae HKY466; 5, K. pneumoniae HKY474. (B) Lanes: M, PFGE marker; A, E. coli HKY154; B, E. coli HKY191; C, E. coli HKY200; D, E. coli HKY215; E, E. coli HKY224; F, E. coli HKY297; G, E. coli HKY315; H, E. coli HKY334; and I, E. coli HKYM68.

Plasmid analyses and Southern hybridization. The plasmid DNA profiles prepared from the five K. pneumoniae isolates and their transconjugants and from two representative E. coli isolates (isolates HKY154 and HKYM68) and their transconjugants are shown in Fig. 3A. One to three large plasmids were apparently present in the five K. pneumoniae isolates, and one large plasmid was transferred to E. coli from each strain in the conjugation experiment. E. coli isolate HKY154 had four plasmids, and identical plasmid profiles were found in seven other E. coli isolates (isolates HKY191, HKY200, HKY215, HKY224, HKY297, HKY315, and HKY334) (data not shown). E. coli HKYM68 harbored three plasmids that were similar to three of the four plasmids found in HKY154. All the E. coli transconjugants carried a single plasmid apparently identical to that transferred from the K. pneumoniae isolates (Fig. 3A and data not shown).

View larger version (87K): [in this window] [in a new window]

FIG. 3. Plasmid profiles and Southern hybridization. (A) Plasmid profiles of clinical isolates and their tranconjugants; (B) hybridization with the probe specific for the CMY-1- and MOX-1-type ß-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1, K. pneumoniae HKY209; 2, E. coli CSH-2/pK209; 3, K. pneumoniae HKY327; 4, E. coli CSH-2/pK327; 5, K. pneumoniae HKY363; 6, E. coli CSH-2/pK363; 7, K. pneumoniae HKY466; 8, E. coli CSH-2/pK466; 9, K. pneumoniae HKY474; 10, E. coli CSH-2/pK474; 11, E. coli HKY154; 12, E. coli CSH-2/pE154; 13, E. coli HKYM68; and 14, E. coli CSH-2/pEM68.

A plasmid of similar size hybridized with the DNA probe specific for the CMY-1-type ß-lactamase genes in all the K. pneumoniae and E. coli isolates and their transconjugants (Fig. 3B). The SacI restriction profiles of the plasmid DNAs from representative transconjugants were very similar to each other (Fig. 4A), and the DNA probe specific for bla_CMY-1-group genes hybridized with a band of about 4.8 kb in size in all cases (Fig. 4B).

View larger version (70K): [in this window] [in a new window]

FIG. 4. Plasmid patterns after restriction enzyme digestion and Southern hybridization. (A) SacI-digested plasmid DNAs prepared from the representative transconjugants; (B) hybridization patterns with the probe specific for CMY-1- and MOX-1-type ß-lactamase gene. Lanes: M, HindIII-digested DNA marker; 1, E. coli CSH-2/pK209; 2, E. coli CSH-2/pK327; 3, E. coli CSH-2/pK363; 4, E. coli CSH-2/pK466; 5, E. coli CSH-2/pK474; 6, E. coli CSH-2/pE154; and 7, E. coli CSH-2/pEM68.

Isoelectric focusing of ß-lactamases. A ß-lactamase band with a pI of >8.45 was detected in all K. pneumoniae and E. coli clinical isolates and their transconjugants but not in E. coli CSH-2 (data not shown). This band likely corresponded to the CMY-9 or CMY-19 ß-lactamase. No band with an acidic pI value was detected in the nine E. coli clinical isolates carrying the bla_TEM gene, likely because of a low level of expression of that gene.

Genetic environments of bla_CMY-9 and bla_CMY-19 genes. The structure of the flanking regions of the bla_CMY-9 gene in E. coli HKYM68 was already reported in a previous study (12). The structure surrounding the bla_CMY genes in the other 13 isolates was identical to that found in HKYM68. Both bla_CMY-9 and bla_CMY-19 were located at the 3' end of a putative transposase gene, orf513. A sul1-type class 1 integron structure consisting of intI1 (an integrase gene), a fused aacA1-orfG gene cassette (responsible for aminoglycoside resistance), qacE1, and sul1 (responsible for trimethoprim-sulfamethoxazole resistance) were found at the 5' end of orf513 (Fig. 5).

View larger version (10K): [in this window] [in a new window]

FIG. 5. Gene organization around blaCMY genes. The blaCMY gene on the conjugative plasmid found in the K. pneumoniae and E. coli clinical isolates located just downstream of orf513 is shown as it was found in our previous study on a CMY-9 producing E. coli HKHM68 (12). Open circle, position of the 59-base element; CS, conserved segment of a class 1 integron. orf513 is speculated to encode a putative transposase, and various antimicrobial resistance genes tend to be integrated just downstream the orf513. The product from the yqgF gene encodes a hypothetical protein very similar to the YqgF identified in Aeromonas hydrophila (EMBL accession no. AJ276030), but the function is unknown.

MICs for CMY-9- or CMY-19-producing E. coli transformants. The MICs of various ß-lactams for CMY-9- or CMY-19-producing E. coli transformants are shown in Table 2. Some notable differences were observed between the MICs of the two strains. The MICs of ampicillin and piperacillin for the CMY-19 producer were higher than those for the CMY-9 producer. Concerning ceftizoxime and cefotaxime, the MICs for the CMY-9 producer were higher than those for the CMY-19 producer, but in the case of ceftazidime, the level of resistance was reversed. The CMY-19 producer showed higher levels of resistance to cefpirome and cefepime than the CMY-9 producer. The MICs of cephamycins, such as cefoxitin, cefmetazole, and cefminox, were higher for the CMY-9 producer than for the CMY-19 producer. A remarkable reduction in the MICs by the addition of a class C ß-lactamase specific inhibitor, 3-aminophenyl boronic acid, was observed with both the CMY-9 and the CMY-19 producers.

View this table: [in this window] [in a new window]

TABLE 2. MICs of ß-lactams for CMY-9-producing and CMY-19-producing E. coli transformants

Kinetic parameters. To purify the CMY-9 and the CMY-19 ß-lactamases, initially, E. coli DH5(pBC-CMY-9) and E. coli DH5(pBC-CMY-19) were cultured in 2 liters of LB broth. However, the yield of purified CMY-19 ß-lactamase was insufficient for the assay of kinetic parameters. Therefore, a pET29a(+) expression vector and an E. coli BL21(DE3) pLysS strain were used for overproduction and purification of that enzyme. The purified enzymes gave a single band on SDS-PAGE with CBB staining that suggested >95% purity (data not shown).

The kinetic parameters of CMY-9 and CMY-19 against selected ß-lactams are shown in Table 3. The hydrolyzing activity (k_cat/K_m) of CMY-19 for penicillins, including ampicillin and piperacillin, were higher than those of CMY-9. Although CMY-9 and CMY-19 had similar k_cat values for cefotaxime, CMY-19 had a 100-fold-higher K_m than CMY-9, resulting in a lower catalytic efficiency for this substrate. Ceftazidime, cefpirome, and cefepime behaved as poor substrates for CMY-9 due to the high K_m values for these agents, while CMY-19 showed different behaviors against these compounds. CMY-19 had a 140-fold-lower K_m against ceftazidime than CMY-9. The k_cat value of CMY-9 for cefepime could not be determined, but CMY-19 measurably hydrolyzed this compound. The hydrolyzing efficiencies (k_cat/K_m values) of CMY-19 against cephamycins such as cefoxitin and cefmetazole were lower than those of CMY-9. Although CMY-19 had a lower K_m against cephamycins than CMY-9, it showed a much lower k_cat against these compounds.

View this table: [in this window] [in a new window]

TABLE 3. Kinetic parameters of CMY-9 and CMY-19

Top Abstract Introduction Materials and Methods Results Discussion References

 
A plasmid-mediated class C ß-lactamase (CMY-1) was first reported in 1989 in a K. pneumoniae isolated in South Korea (8). Subsequently, several variants of that enzyme, such as MOX-1 (16), CMY-8 (33), CMY-9 (12), CMY-10 (20), and CMY-11 (21), have been identified, mainly in East Asian countries, including Taiwan and Japan. The dissemination of CMY-10- and CMY-11-producing isolates of the family Enterobacteriaceae was also reported in Korea (18, 19). In the present study, CMY-type ß-lactamase-producing K. pneumoniae and E. coli clinical isolates from a Japanese general hospital were investigated. Through PFGE analysis, it was found that the five K. pneumoniae isolates had little genetic relatedness to each other, while the nine E. coli isolates belonged to the same clonal lineage. Interestingly, plasmid analysis showed that all 14 isolates harbored a very similar conjugal plasmid that encodes a CMY-type ß-lactamase which was either CMY-9 or CMY-19, a variant that differs from CMY-9 by a single amino acid substitution (I292S). Since the flanking structures of the bla_CMY genes were identical in all plasmids, it is probable that one conjugal plasmid carrying bla_CMY-9 was horizontally transferred to E. coli, K. pneumoniae, and then an E. coli clone and various K. pneumoniae strains harboring the bla_CMY gene-bearing plasmids might have spread in the hospital.

ß-Lactamases can modify their substrate specificity through a single amino acid substitution (30). CMY-19 had a single amino acid substitution, I292S, near the H-10 helix domain, compared with the sequence of CMY-9 (Fig. 1). Indeed, a serine residue at the same amino acid position was found in all the FOX-type enzymes (15, 26), including CAV-1 (14), and also in CMY-11 (21), as shown Fig. 1; but no peculiar behavior against cefepime was documented with those enzymes. Through the I292S substitution, CMY-19 would have developed extended substrate specificity against cefepime and cefpirome, as well as ampicillin, piperacillin, cephaloridine, and ceftazidime, compared with that of CMY-9, although the hydrolyzing activities against ceftizoxime, cefotaxime, and cephamycins were impaired. The expansion of hydrolyzing activity against cefepime found in CMY-19 was a most remarkable property because cefepime is generally stable against AmpC ß-lactamases (11). Similar developments of extended hydrolyzing activity against cefepime through amino acid substitutions or deletions adjacent to the H-10 helix have been observed in several chromosomally encoded AmpC ß-lactamases, such as the AmpC of Serratia marcescens that lacks four amino acids at positions 293 to 296 (22), an AmpC of E. coli that lacks three amino acids at positions 286 to 288 (13), an AmpC of Enterobacter cloacae that lacks six amino acids at positions 289 to 294 (6), and an AmpC of Enteroacter aerogenes that acquired an L293P substitution (5). Furthermore, Barlow and Hall reported on the in vitro selection of CMY-2 ß-lactamase variants with several amino acid substitutions, including replacements at positions 292, 293, 294, 296, and 298, which demonstrated increased resistance to cefepime (4). From our previous molecular modeling analyses (13), it was found that the expansion of an open space in the vicinity of the R-2 side chain of ceftazidime or cefepime through the deletion of tripeptides in the H-10 helix of E. coli AmpC (Fig. 1) played a crucial role in the acquisition of the greater hydrolyzing activity against those agents possessing a bulky R side chain. Although the actual mechanism for the higher cefepime-hydrolyzing activity in CMY-19 compared to that in CMY-9 has not been elucidated, a similar molecular distortion at the active center of the enzyme might well have occurred in CMY-19. This speculation would be substantiated by molecular modeling and X-ray crystallographic analyses.

 
We are grateful to Kumiko Kai and Fusako Yokokawa for technical assistance.

This work was supported by the Ministry of Health, Labor and Welfare of Japan (grants H15-Shinkou-9 and H15-Shinkou-10). The research activity of J. Wachino was supported by a Scholarship for Young Scientists provided by the Japan Society for the Promotion of Science.

 
* Corresponding author. Mailing address: Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashi-Murayama, Tokyo 208-0011, Japan. Phone: 81-42-561-0771, ext. 500. Fax: 81-42-561-7173. E-mail: yarakawa{at}nih.go.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Ambler, R. P. 1980. The structure of ß-lactamases. Philos. Trans. R. Soc. London B Biol. Sci. 289:321-331.[Abstract/Free Full Text]
2
2. Arakawa, Y., N. Shibata, K. Shibayama, H. Kurokawa, T. Yagi, H. Fujiwara, and M. Goto. 2000. Convenient test for screening metallo-ß-lactamase-producing gram-negative bacteria by using thiol compounds. J. Clin. Microbiol. 38:40-43.[Abstract/Free Full Text]
3
3. Armand-Lefevre, L., V. Leflon-Guibout, J. Bredin, F. Barguellil, A. Amor, J. M. Pages, and M. H. Nicolas-Chanoine. 2003. Imipenem resistance in Salmonella enterica serovar Wien related to porin loss and CMY-4 ß-lactamase production. Antimicrob. Agents Chemother. 47:1165-1168.[Abstract/Free Full Text]
4
4. Barlow, M., and B. G. Hall. 2003. Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC ß-lactamase. Genetics 164:23-29.[Abstract/Free Full Text]
5
5. Barnaud, G., Y. Benzerara, J. Gravisse, L. Raskine, M. J. Sanson-Le Pors, R. Labia, and G. Arlet. 2004. Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate. Antimicrob. Agents Chemother. 48:1040-1042.[Abstract/Free Full Text]
6
6. Barnaud, G., R. Labia, L. Raskine, M. J. Sanson-Le Pors, A. Philippon, and G. Arlet. 2001. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS Microbiol. Lett. 195:185-190.[CrossRef][Medline]
7
7. Bauernfeind, A., I. Stemplinger, R. Jungwirth, R. Wilhelm, and Y. Chong. 1996. Comparative characterization of the cephamycinase bla_CMY-1 gene and its relationship with other ß-lactamase genes. Antimicrob. Agents Chemother. 40:1926-1930.[Abstract]
8
8. Bauernfeind, A., Y. Chong, and S. Schweighart. 1989. Extended broad-spectrum ß-lactamase in Klebsiella pneumoniae including resistance to cephamycins. Infection 17:316-321.[CrossRef][Medline]
9
9. Bradford, P. A., C. Urban, N. Mariano, S. J. Projan, J. J. Rahal, and K. Bush. 1997. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrob. Agents Chemother. 41:563-569.[Abstract]
10
10. Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211-1233.[Medline]
11
11. Chapman, T. M., and C. M. Perry. 2003. Cefepime: a review of its use in the management of hospitalized patients with pneumonia. Am. J. Respir. Med. 2:75-107.[Medline]
12
12. Doi, Y., N. Shibata, K. Shibayama, K. Kamachi, H. Kurokawa, K. Yokoyama, T. Yagi, and Y. Arakawa. 2002. Characterization of a novel plasmid-mediated cephalosporinase (CMY-9) and its genetic environment in an Escherichia coli clinical isolate. Antimicrob. Agents Chemother. 46:2427-2434.[Abstract/Free Full Text]
13
13. Doi, Y., J. Wachino, M. Ishiguro, H. Kurokawa, K. Yamane, N. Shibata, K. Shibayama, K. Yokoyama, H. Kato, T. Yagi, and Y. Arakawa. 2004. Inhibitor-sensitive AmpC ß-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins. Antimicrob. Agents Chemother. 48:2652-2658.[Abstract/Free Full Text]
14
14. Fosse, T., C. Giraud-Morin, I. Madinier, and R. Labia. 2003. Sequence analysis and biochemical characterisation of chromosomal CAV-1 (Aeromonas caviae), the parental cephalosporinase of plasmid-mediated AmpC ‘FOX’ cluster. FEMS Microbiol. Lett. 222:93-98.[CrossRef][Medline]
15
15. Gonzalez Leiza, M., J. C. Perez-Diaz, J. Ayala, J. M. Casellas, J. Martinez-Beltran, K. Bush, and F. Baquero. 1994. Gene sequence and biochemical characterization of FOX-1 from Klebsiella pneumoniae, a new AmpC-type plasmid-mediated ß-lactamase with two molecular variants. Antimicrob. Agents Chemother. 38:2150-2157.[Abstract/Free Full Text]
16
16. Horii, T., Y. Arakawa, M. Ohta, S. Ichiyama, R. Wacharotayankun, and N. Kato. 1993. Plasmid-mediated AmpC-type ß-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum ß-lactams, including moxalactam. Antimicrob. Agents Chemother. 37:984-990.[Abstract/Free Full Text]
17
17. Kado, C. I., and S. T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365-1373.[Abstract/Free Full Text]
18
18. Kim, J. Y., J. S. Song, S. H. Bak, Y. E. Cho, D. W. Kim, S. H. Jeong, Y. M. Park, K. J. Lee, and S. H. Lee. 2004. Dissemination of Escherichia coli producing AmpC-type ß-lactamase (CMY-11) in Korea. Int. J. Antimicrob. Agents 24:320-326.[CrossRef][Medline]
19
19. Lee, J. H., H. I. Jung, J. H. Jung, J. S. Park, J. B. Ahn, S. H. Jeong, B. C. Jeong, and S. H. Lee. 2004. Dissemination of transferable AmpC-type ß-lactamase (CMY-10) in a Korean hospital. Microb. Drug Resist. 10:224-230.[Medline]
20
20. Lee, S. H., S. H. Jeong, and Y. M. Park. 2003. Characterization of bla_CMY-10, a novel plasmid-encoded AmpC-type ß-lactamase gene in a clinical isolate of Enterobacter aerogenes. J. Appl. Microbiol. 95:744-752.[CrossRef][Medline]
21
21. Lee, S. H., J. Y. Kim, G. S. Lee, S. H. Cheon, Y. J. An, S. H. Jeong, and K. J. Lee. 2002. Characterization of bla_CMY-11, an AmpC-type plasmid-mediated ß-lactamase gene in a Korean clinical isolate of Escherichia coli. J. Antimicrob. Chemother. 49:269-273.[Abstract/Free Full Text]
22
22. Mammeri, H., L. Poirel, P. Bemer, H. Drugeon, and P. Nordmann. 2004. Resistance to cefepime and cefpirome due to a 4-amino-acid deletion in the chromosome-encoded AmpC ß-lactamase of a Serratia marcescens clinical isolate. Antimicrob. Agents Chemother. 48:716-720.[Abstract/Free Full Text]
23
23. Nakano, R., R. Okamoto, Y. Nakano, K. Kaneko, N. Okitsu, Y. Hosaka, and M. Inoue. 2004. CFE-1, a novel plasmid-encoded AmpC ß-lactamase with an ampR gene originating from Citrobacter freundii. Antimicrob. Agents Chemother. 48:1151-1158.[Abstract/Free Full Text]
24
24. National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Document M7-A5. National Committee for Clinical Laboratory Standards, Wayne, Pa.
25
25. Philippon, A., G. Arlet, and G. A. Jacoby. 2002. Plasmid-determined AmpC-type ß-lactamases. Antimicrob. Agents Chemother. 46:1-11.[Free Full Text]
26
26. Queenan, A. M., S. Jenkins, and K. Bush. 2001. Cloning and biochemical characterization of FOX-5, an AmpC-type plasmid-encoded ß-lactamase from a New York City Klebsiella pneumoniae clinical isolate. Antimicrob. Agents Chemother. 45:3189-3194.[Abstract/Free Full Text]
27
27. Sanders, C. C. 1987. Chromosomal cephalosporinases responsible for multiple resistance to newer ß-lactam antibiotics. Annu. Rev. Microbiol. 41:573-593.[CrossRef][Medline]
28
28. Shibata, N., Y. Doi, K. Yamane, T. Yagi, H. Kurokawa, K. Shibayama, H. Kato, K. Kai, and Y. Arakawa. 2003. PCR typing of genetic determinants for metallo-ß-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J. Clin. Microbiol. 41:5407-5413.[Abstract/Free Full Text]
29
29. Stapleton, P. D., K. P. Shannon, and G. L. French. 1999. Carbapenem resistance in Escherichia coli associated with plasmid-determined CMY-4 ß-lactamase production and loss of an outer membrane protein. Antimicrob. Agents Chemother. 43:1206-1210.[Abstract/Free Full Text]
30
30. Wachino, J., Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, and Y. Arakawa. 2004. Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A ß-lactamase, GES-4, possessing a single G170S substitution in the omega-loop. Antimicrob. Agents Chemother. 48:2905-2910.[Abstract/Free Full Text]
31
31. Wachino, J., Y. Doi, K. Yamane, N. Shibata, T. Yagi, T. Kubota, H. Ito, and Y. Arakawa. 2004. Nosocomial spread of ceftazidime-resistant Klebsiella pneumoniae strains producing a novel class a ß-lactamase, GES-3, in a neonatal intensive care unit in Japan. Antimicrob. Agents Chemother. 48:1960-1967.[Abstract/Free Full Text]
32
32. Yagi, T., J. Wachino, H. Kurokawa, K. Yamane, Y. Doi, N. Shibata, H. Kato, K. Shibayama, and Y. Arakawa. 2004. Practical methods for identification of class C ß-lactamase-producing Klebsiella pneumoniae and Escherichia coli using boronic acid compounds. J. Clin. Microbiol. 43:2551-2558.
33
33. Yan, J. J., S. M. Wu, S. H. Tsai, J. J. Wu, and I. J. Su. 2000. Prevalence of SHV-12 among clinical isolates of Klebsiella pneumoniae producing extended-spectrum ß-lactamases and identification of a novel AmpC enzyme (CMY-8) in southern Taiwan. Antimicrob. Agents Chemother. 44:1438-1442.[Abstract/Free Full Text]

---

Antimicrobial Agents and Chemotherapy, February 2006, p. 534-541, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.534-541.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Doi, Y., Paterson, D. L., Adams-Haduch, J. M., Sidjabat, H. E., O'Keefe, A., Endimiani, A., Bonomo, R. A. (2009). Reduced Susceptibility to Cefepime among Escherichia coli Clinical Isolates Producing Novel Variants of CMY-2 {beta}-Lactamase. Antimicrob. Agents Chemother. 53: 3159-3161 [Abstract] [Full Text]  
• Tenover, F. C., Emery, S. L., Spiegel, C. A., Bradford, P. A., Eells, S., Endimiani, A., Bonomo, R. A., McGowan, J. E. Jr. (2009). Identification of Plasmid-Mediated AmpC {beta}-Lactamases in Escherichia coli, Klebsiella spp., and Proteus Species Can Potentially Improve Reporting of Cephalosporin Susceptibility Testing Results. J. Clin. Microbiol. 47: 294-299 [Abstract] [Full Text]  
• Jacoby, G. A. (2009). AmpC {beta}-Lactamases. Clin. Microbiol. Rev. 22: 161-182 [Abstract] [Full Text]  
• Nagano, Y., Nagano, N., Wachino, J.-i., Ishikawa, K., Arakawa, Y. (2009). Novel Chimeric {beta}-Lactamase CTX-M-64, a Hybrid of CTX-M-15-Like and CTX-M-14 {beta}-Lactamases, Found in a Shigella sonnei Strain Resistant to Various Oxyimino-Cephalosporins, Including Ceftazidime. Antimicrob. Agents Chemother. 53: 69-74 [Abstract] [Full Text]  
• Sohn, S. G., Lee, J. J., Sohn, E. S., Kang, L.-W., Lee, S. H. (2008). Comment on: Extension of the hydrolysis spectrum of AmpC {beta}-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. J Antimicrob Chemother 61: 965-966 [Full Text]  
• Vimont, S., Aubert, D., Mazoit, J.-X., Poirel, L., Nordmann, P. (2007). Broad-spectrum {beta}-lactams for treating experimental peritonitis in mice due to Escherichia coli producing plasmid-encoded cephalosporinases. J Antimicrob Chemother 60: 1045-1050 [Abstract] [Full Text]  
• Hee Lee, S., Lee, J. H., Heo, M. J., Bae, I. K., Jeong, S. H., Cha, S.-S., Wachino, J.-i., Arakawa, Y. (2007). Exact Location of the Region Responsible for the Extended Substrate Spectrum in Class C {beta}-Lactamases. Antimicrob. Agents Chemother. 51: 3778-3779 [Full Text]  

This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wachino, J.-i. Articles by Arakawa, Y. Search for Related Content PubMed PubMed Citation Articles by Wachino, J.-i. Articles by Arakawa, Y.