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Antimicrobial Agents and Chemotherapy, August 2004, p. 2905-2910, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.2905-2910.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Characterization of a Cephamycin-Hydrolyzing and Inhibitor-Resistant Class A ß-Lactamase, GES-4, Possessing a Single G170S Substitution in the -Loop

Jun-ichi Wachino,^1,2 Yohei Doi,¹ Kunikazu Yamane,¹ Naohiro Shibata,¹ Tetsuya Yagi,¹ Takako Kubota,³ and Yoshichika Arakawa^1*

Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo,¹ Central Clinical Laboratory, Kagoshima Municipal Hospital, Kagoshima,³ Program in Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan²

Received 26 August 2003/ Returned for modification 19 November 2003/ Accepted 5 April 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type ß-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new ß-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 µg/ml) and ß-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 ± 1.7 µM). The GES-4 enzyme had a single G170S substitution in the -loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of ß-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla_GES-4 genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla_GES-4 genes found in strain KG502 were almost identical to that of bla_GES-3 in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla_GES-4 gene found in strain KG502 might well emerge from a point mutation in the bla_GES-3 gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over the past decade, a number of new plasmid-mediated ß-lactamases with wide substrate specificities have appeared mainly in gram-negative bacilli belonging to the family Enterobacteriaceae (11, 19, 20, 27). In particular, the emergence of bacteria producing TEM- and SHV-derived extended-spectrum ß-lactamases (ESBLs) has made chemotherapy for bacterial infections more complex than ever (13). Furthermore, non-TEM- and non-SHV-type ESBLs, such as the CTX-M-type (23, 31), GES-type (8, 21, 25, 28, 29), and VEB-type (3, 22) ß-lactamases, have also been identified in these gram-negative bacilli. Generally, the ß-lactamases described above are often plasmid encoded and can hydrolyze oximino-cephalosporins and monobactams as well as penicillins but not 7--methoxy-cephalosporins, the so-called cephamycins. Carbapenems are also very stable against these enzymes. Therefore, at present cephamycins and carbapenems are potent agents for the treatment of infections caused by the gram-negative bacilli that produce these new class A ß-lactamases with wide substrate specificities. Among the various ß-lactamase genes described above, the genes encoding the GES-type ß-lactamases as well as the VEB-type ß-lactamases are often located in integrons as gene cassettes (8, 17, 21, 22, 25, 29). Integrons have been described to play a sophisticated role in the accumulation and expression of genes responsible for antibiotic resistance as well as their dissemination among gram-negative bacilli (9, 10).

Among the GES-type ß-lactamases, GES-1 was first reported from a Klebsiella pneumoniae clinical isolate in France in 1998 (21); and then two other GES-type ß-lactamases, IBC-1 and GES-2, were found in Enterobacter cloacae and Pseudomonas aeruginosa, respectively (8, 25). GES-2 has an amino acid substitution (glycine to asparagine at position 170) compared to the sequence of GES-1 and shows a higher imipenem-hydrolyzing activity than GES-1.

We found that the high-level ceftazidime resistance of six genetically related K. pneumoniae clinical strains, which had been isolated from a neonatal intensive care unit (NICU) over a 1-year period, depended on the production of GES-type ß-lactamases, and one of the six isolates was found to produce the GES-3 ß-lactamase (30). The bla_GES-3 gene encoding GES-3 was located as a gene cassette in a class 1 integron, as has been observed for the GES-type ß-lactamase genes found in Europe. GES-3 production does not affect the level of cephamycin resistance in the Escherichia coli host, as has been reported for the other Ambler class A ß-lactamases, including ESBLs. However, the levels of resistance to cephamycins varied widely among the six GES-type ß-lactamase-producing strains. Among these, the highest MICs of the carbapenems as well as the cephamycins were seen for strain KG502 (30), which also showed an inhibitor resistance phenotype. The goal of this study was to elucidate the molecular mechanism responsible for resistance to cephamycins and carbapenems in strain KG502, as well as its inhibitor-resistant nature.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. K. pneumoniae strain KG502 was isolated in May 2002 from the pus of a neonate under treatment in the NICU of a general hospital in Japan. This strain was resistant to oximino-cephalosporins and cephamycins. GES-3-producing strain KG525 was isolated in the same NICU where strain KG502 was isolated.

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TABLE 1. Bacterial strains and plasmids used in this study

Susceptibility testing. MICs were determined by the agar dilution method with Mueller-Hinton agar (Becton Dickinson, Cockeysville, Md.), according to the guidelines in National Committee for Clinical Laboratory Standards document M7-A5 (18). E. coli ATCC 25922 and ATCC 35218 were purchased from the American Type Culture Collection (ATCC) and served as control strains for MIC determinations. The double-disk synergy test for the detection of ESBL production and an inhibitory test with thiol compounds for the detection of metallo-ß-lactamase producers were carried out by the methods described elsewhere (1, 6, 12), with the modification that 2-mercaptopropionic acid was replaced with sodium mercaptoacetic acid.

Transfer of ß-lactam resistance genes. Conjugation experiments were performed by the filter mating method with rifampin- and nalidixic acid-resistant E. coli CSH-2 as the recipient. Transconjugants were detected on Luria-Bertani (LB) agar supplemented with rifampin (100 µg/ml), nalidixic acid (100 µg/ml), and either ceftazidime (4 µg/ml) or cefminox (2 µg/ml). Transformation of E. coli XL1-Blue with the large plasmids of the parental strain K. pneumoniae KG502 was performed by electroporation. Transformants were selected on LB agar containing ceftazidime (4 µg/ml) or cefminox (2 µg/ml).

PCR amplification, cloning, and sequencing of ß-lactamase gene. To amplify the bla_GES gene, PCR was performed with the primers under the conditions described elsewhere (30). The cloning experiment was carried out as follows: total DNA prepared from strain K. pneumoniae KG502 was digested with BamHI, and the resultant fragments were ligated to vector pBCSK+ (Stratagene, La Jolla, Calif.), which had been digested with the same enzyme. Transformants carrying recombinant plasmids were selected on LB agar plates containing chloramphenicol (30 µg/ml) and either ceftazidime (4 µg/ml) or cefminox (2 µg/ml). Both strands of the DNA fragments inserted into the recombinant plasmids (pKGL502 and pKGS502) were sequenced.

Purification of ß-lactamase. ß-Lactamases were purified by exactly the same protocol described elsewhere (30). In brief, ß-lactamases were overproduced with the pET system, extracted by use of a French press, and cleared by ultracentrifugation. After ultracentrifugation the supernatant was loaded onto a HiLoad 16/60 Superdex 200 and anion-exchange Hitrap Q HP column (Pharmacia Biotech, Uppsala, Sweden). The purity of the enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

ß-Lactamase assay. Substrate hydrolyses by GES-4 and GES-3 were assayed at 30°C in phosphate buffer (50 mM; pH 7.0) by use of an autospectrophotometer (V-550; Nihon Bunko Ltd., Tokyo, Japan). The molar extinction coefficients ( values) used were as follows: for benzylpenicillin (232 nm), 1.077 mM^–1 cm^–1; for ampicillin (235 nm), 1.121 mM^–1 cm^–1; for cephaloridine (300 nm), 0.384 mM^–1 cm^–1; for cefotaxime (264 nm), 5.725 mM^–1 cm^–1; for ceftazidime (274 nm), 6.123 mM^–1 cm^–1; for cefpirome (290 nm), 4.057 mM^–1 cm^–1; for cefoxitin (293 nm), 0.325 mM^–1 cm^–1; for cefminox (298 nm), 1.878 mM^–1 cm^–1; for imipenem (297 nm), 8.061 mM^–1 cm^–1; and for aztreonam (315 nm), 0.68 mM^–1 cm^–1. Fifty percent inhibitory concentrations (IC₅₀s) were determined with benzylpenicillin as the substrate and the inhibitors clavulanic acid, sulbactam, tazobactam, and imipenem. Purified enzyme and various concentrations of these inhibitors were preincubated in 50 mM phosphate buffer (pH 7.0) at 30°C for 5 min. Purified GES-4 and GES-3 ß-lactamases and nonpurified extracts of 50-ml cultures of strain KG502 were subjected to isoelectric focusing (IEF) analysis with an Immmobiline Drystrip (pH 3 to 10; Pharmacia Biotech) and an IPGphor electrophoresis system (Pharmacia Biotech).

Nucleotide sequence accession numbers. The nucleotide sequences described in this work appear in the GenBank nucleotide database under accession numbers AB116260 and AB116723.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Properties of K. pneumoniae isolate KG502. K. pneumoniae KG502 was isolated from the pus of a neonate in May 2002. This strain was one of the six GES-type ß-lactamase-producing strains that we reported previously (30). Strain KG502 exhibited resistance to oximino-cephalosporins and the cephamycins and intermediate susceptibility to carbapenems. No synergy between an amoxicillin-clavulanic acid disk and a ceftazidime and/or a cefotaxime disk was detectable against this strain. The lack of production of metallo-ß-lactamases was also suggested by the results of inhibition testing with sodium mercaptoacetic acid. Preliminary PCR detection of some class A ß-lactamase and metallo-ß-lactamase genes was performed as we reported in our previous study (30), and all PCRs gave negative results.

Transfer of ß-lactam resistance by conjugation and transformation. Our previous Southern hybridization experiment with a digoxigenin-labeled bla_GES-specific probe demonstrated that the GES-type ß-lactamase genes of strain KG502 are located on two distinct plasmids. Therefore, we performed conjugation by filter mating in an attempt to transfer these plasmids to E. coli CSH-2, as well as electroporation to introduce them directly into E. coli XL1-Blue. However, the transfer of these plasmids into E. coli was unsuccessful, despite repeated attempts.

Cloning and sequencing of the ß-lactamase gene. Sequencing of the DNAs of the PCR products obtained with the primers specific for the GES-type ß-lactamase gene revealed the presence of a variant of the bla_GES-3 gene in strain KG502. Cloning was performed by standard procedures to determine the entire nucleotide sequences of this new gene. Two distinct recombinant plasmids, one of which carried a 6.6-kb BamHI fragment and the other of which carried a 6.0-kb BamHI fragment, were obtained independently. The nucleotide sequences of both genetic determinants for ß-lactam resistance were the same and differed by a glycine (G)-to-alanine (A) mutation at position 509 compared with the sequence of bla_GES-3, so they were designated bla_GES-4. The deduced amino acid sequence of GES-4 had an amino acid substitution of G to serine (S) at position 170 (G170S) within the -loop region of the Ambler class A ß-lactamase compared with the sequence of GES-3 (Fig. 1). Among the GES-type ß-lactamases, a similar amino acid substitution was reported at position 170, G to asparagine (N), leading to the conversion from GES-1 to GES-2 (25).

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FIG. 1. Amino acid alignments of GES-4, GES-3, GES-2, and GES-1 ß-lactamases. Hyphens indicate identical amino acids, and the -loop region of ß-lactamase is underlined.

Sequencing of bla_GES-4 flanking region. The inserts in recombinant plasmids pKGL502 and pKGS502 were sequenced, which revealed that both fragments commonly contained bla_GES-4 gene in the class 1 integron separately, followed by an aacA1-orfG fused gene, as the first and second gene cassettes, respectively. The integron in pKGL502 differed from that in pKGS502, in that a third gene cassette, orfA, was present (Fig. 2). The product encoded by orfA had no significant homology with any other known protein at the amino acid sequence level, so the function of the product could not be presumed. Moreover, the backbone genetic structure surrounding the integron containing the bla_GES-4 gene from strain KG502 was otherwise identical to that surrounding the integron containing the bla_GES-3 gene from strain KG525, except that it lacked the outer 128-bp nucleotide sequences, including the 25-bp terminal repeat (IRt) at the left end of IS6100, as shown in Fig. 2.

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FIG. 2. Schematic comparison of the genetic environments of three class 1 integrons mediating blaGES-4 on pKGL502 and pKGS502 and blaGES-3 on pKGB525. Open circles represent the positions of the 59-base element. CS, conserved segment.

Susceptibilities to various ß-lactams. The MICs of ß-lactams for K. pneumoniae strain KG502 and E. coli XL1-Blue harboring recombinant plasmid pKGL502 are listed in Table 2. GES-4 ß-lactamase-producing strain KG502 exhibited resistance to cefminox, moxalactam, and cefmetazole and intermediate susceptibility to imipenem and meropenem, whereas GES-3-producing strain KG525 was susceptible to all these agents. These resistance trends were also observed in each of the E. coli clones harboring pKGL502 or pKGB525, but the overall resistance levels of the clones were lower than those of the parent strains.

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TABLE 2. MICs of ß-lactams

Two notable differences were detected between the MICs for a GES-4-producing E. coli clone (pKGL502) and those for a GES-3-producing E. coli clone (pKGB525), expressed under the same promoters located within intI1. One was a difference in the levels of resistance to cephamycins. The MICs of cephamycins, such as cefminox, cefoxitin, moxalactam, and cefmetazole, were much higher for the GES-4-producing clone than the GES-3-producing clone. In addition, the meropenem MIC for the GES-4 producer was 16-fold higher than that for the GES-3 producer.

The other major differences were the inhibition profiles obtained when the ß-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam were added. The MICs of ampicillin, amoxicillin, piperacillin, ceftazidime, and cefotaxime for the GES-4-producing E. coli clone were decreased a maximum of only 8-fold in the presence of ß-lactamase inhibitors, whereas those for the GES-3-producing E. coli clone decreased at least 32- to 512-fold.

IEF analysis. IEF analysis of the crude extract of parent strain KG502 revealed the presence of two major bands with ß-lactamase activities corresponding to pIs of 6.9 and 7.6, respectively. The band with a pI of 7.6 was likely the chromosomally encoded SHV-type ß-lactamase of K. pneumoniae. The estimated pI of 6.9 was identical to those of the purified GES-3 and GES-4 enzymes.

Kinetic studies. The kinetic parameters of the GES-4 and GES-3 ß-lactamases for representative ß-lactams are given in Table 3. The hydrolyzing efficiencies (k_cat/K_m) of GES-4 for the penicillins were about twice as high as those of GES-3, although GES-2 showed less efficient hydrolysis but lower K_m values for cephaloridine and penicillin. On the other hand, GES-3 hydrolyzed ceftazidime and cefotaxime more efficiently than GES-4 did. GES-4 measurably hydrolyzed cefoxitin, cefminox, and imipenem, which accounted for the increases in the MICs of these agents for the clone harboring pKGL502, but no measurable hydrolysis of these agents as substrates by GES-3 was observed under the same experimental conditions used in the present study. No measurable hydrolysis was observed for aztreonam as the substrate with each type of ß-lactamase. The IC₅₀s measured with benzylpenicillin as the substrate are listed in Table 4. GES-2 was reported to be inhibited by lower concentrations of clavulanic acid and tazobactam; but GES-4 was inhibited 10-fold less by clavulanic acid, 16-fold less by sulbactam, 8-fold less by tazobactam, and 21-fold less by imipenem than GES-3 was. These results corroborate the inhibitor-resistant nature of GES-4.

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TABLE 3. Kinetic parameters of GES-4, GES-3, and GES-2

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TABLE 4. Inhibition profiles of GES-type ß-lactamases

Top Abstract Introduction Materials and Methods Results Discussion References

 
GES-4 had a single G170S substitution within the -loop region of class A ß-lactamases compared with the sequence of GES-3. Replacement of the side chain (H) of the glycine residue with that (CH₂OH) of the serine residue may indeed contribute to the acceleration of cephamycin hydrolysis as well as the inhibitor resistance profile. The GES-2 ß-lactamase, identified as a variant of GES-1, had a substitution from glycine to asparagine at position 170, which is the same position leading to the conversion from GES-3 to GES-4. In comparison with GES-1, GES-2 showed an extended substrate specificity for imipenem and a lower affinity for ß-lactamase inhibitors (25), as was seen with GES-4. However, the obvious increases in the MICs of cephamycins and meropenem seen for GES-4 were not detected for GES-2. These findings suggest that a single amino acid substitution at position 170, the center of the -loop region, would play a key role in the expansion of the substrate specificities among GES-type ß-lactamases. To elucidate the nature of GES-4, molecular modeling analysis as well as X-ray crystallographic analysis will be undertaken in the next study.

Although amino acid substitutions in the -loop region, which influence hydrolyzing activities against oximino-cephalosporins and carbapenems, have also been observed in several class A ß-lactamases, such as those of the TEM type (5, 16), SHV type (2, 15), CTX-M type (23), and GES type (25). Disruption of the salt bridge between R164 and D179 was suggested to be mainly involved in the expansion of substrate specificity for oxyimino-cephalosporins in these enzymes. However, substitutions resulting in increased cephamycin resistance have not been reported in class A ß-lactamases so far. To our knowledge, this is the first report of a class A ß-lactamase with cephamycin-hydrolyzing ability as a result of a single amino acid substitution in the center of the -loop region. Poyart et al. (26) also reported a similar phenomenon in a TEM-type ß-lactamase (TEM-52), in which significant decreases in vitro susceptibilities to some cephamycins were not due to an amino acid substitution in the -loop region. The same investigators reported, however, that the combination of three amino acid substitutions E104K, M182Y, and G238S (on the basis of the sequence of TEM-1) in TEM-25 was responsible for the elevated MICs of moxalactam and cefotetan.

The G170S substitution found in the GES-4 ß-lactamase affected not only cephamycin and carbapenem resistance but also inhibitor resistance. The IC₅₀s of clavulanic acid, sulbactam, tazobactam, and imipenem for GES-4 were considerably higher than those of GES-3. Since 1990 IRT ß-lactamases derived from TEM-type ß-lactamases have been reported to be inhibitor resistance class A ß-lactamases. The IRT ß-lactamases differ from parental enzyme TEM-1 or TEM-2 by several amino acid substitutions at different locations. The IC₅₀s of clavulanic acid and tazobactam for GES-4 (15.2 and 1.43 µM, respectively) were similar to those of some IRT ß-lactamases, including IRT-7 (23 and 0.9 µM, respectively), IRT-8 (25 and 1 µM, respectively), and IRT-14 (22.5 and 1.48 µM, respectively), while the IC₅₀ of sulbactam for GES-4 was much lower than those for IRTs (4). GES-4 seems to be a very characteristic enzyme, because it has a strong inhibitor-resistant nature like IRT enzymes, while it maintains the capacity to hydrolyze cephamycins and carbapenems.

As with the other bla_GES genes, the bla_GES-4 gene was located in the class 1 integron as a gene cassette. Strain KG502 was unique, in that it possessed two distinct class 1 integrons which carried similar gene cassette configurations, including the bla_GES-4 gene cassette. The coexistence of class 1 integrons with similar gene cassette arrays might result from the duplication of a region containing one original class 1 integron by mobile elements, such as transposons in strain KG502. It was speculated that in this strain a region containing one original class 1 integron with the bla_GES-4, aacA1-orfG, and orfA gene cassettes was first duplicated in the bacterium. Next, one of the class 1 integrons might have excised the orfA gene cassette by a site-specific recombination mechanism catalyzed by some recombinases, including integrases or transposases. Consequently, strain KG502 might have come to have two class 1 integrons with very similar backbone structures.

The entire genetic structure of the flanking region containing bla_GES-4 on pKGL502 was almost identical to that containing bla_GES-3 on pKGB525. Taken together with the facts that both the bla_GES-3 and the bla_GES-4 genes were found in genetically related K. pneumoniae strains and that the genetic environments of these two genes are almost the same, it is probable that the bla_GES-4 gene emerged from the point mutation in the bla_GES-3 gene under conditions of antibiotic stress in order to acquire resistance to additional groups of drugs, i.e., the cephamycins and carbapenems.

We characterized here for the first time a novel class A ß-lactamase, GES-4, which acquired extended substrate specificity for the cephamycins through a single amino acid substitution within the -loop region. This finding indicates that ß-lactamases which are capable of hydrolyzing cephamycins are not limited to the Ambler class B and class C ß-lactamases. The emergence of a cephamycin-hydrolyzing class A ß-lactamase might complicate treatment in clinical settings, because cephamycins have generally been considered stable to class A ß-lactamases and to retain good efficacies for the treatment of infectious diseases caused by organisms producing class A ß-lactamases. Moreover, the inhibitor resistance of the GES-4 ß-lactamase may introduce confusion during the routine laboratory detection of class A ß-lactamase-producing strains, including ESBL producers. The much higher IC₅₀ of clavulanic acid for GES-4 hampered the detection of GES-4-producing clinical isolates by conventional double-disk synergy testing.

Since GES-type ß-lactamase-producing gram-negative bacteria have been identified worldwide and nosocomial outbreaks caused by these microorganisms have been reported worldwide (7, 14, 24), due consideration must be given to the possible emergence of variants of GES-type ß-lactamases like GES-4 which have acquired several amino acid substitutions to expand their substrate specificities to cope with the extensive use of broad-spectrum ß-lactams in clinical settings.

 
We are grateful to Kumiko Kai for technical assistance.

This work was supported by grants H12-Shinko-19, H12-Shinko-20, H15-Shinko-9, and H15-Shinko-10 from the Ministry of Health, Labor and Welfare of Japan.

 
* 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.

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Antimicrobial Agents and Chemotherapy, August 2004, p. 2905-2910, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.2905-2910.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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