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Antimicrobial Agents and Chemotherapy, February 2005, p. 600-605, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.600-605.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel SHV-Derived Extended-Spectrum ß-Lactamase, SHV-57, That Confers Resistance to Ceftazidime but Not Cefazolin

Ling Ma,¹ Jimena Alba,² Feng-Yee Chang,³ Masaji Ishiguro,⁴ Keizo Yamaguchi,² L. K. Siu,^1* and Yoshikazu Ishii²

Division of Clinical Research, National Health Research Institutes,¹ Division of Infectious Disease and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan,³ Department of Microbiology, Toho University School of Medicine, Tokyo,² Suntory Institute for Bioorganic Research, Shimamoto, Osaka, Japan⁴

Received 14 April 2004/ Returned for modification 19 June 2004/ Accepted 3 October 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
A new SHV-derived extended-spectrum ß-lactamase, SHV-57, that confers high-level resistance to ceftazidime but not cefotaxime or cefazolin was identified from a national surveillance study conducted in Taiwan in 1998. An Escherichia coli isolate resistant to ampicillin, cephalothin, and ceftazidime but sensitive to cefoxitin, ceftriaxone, cefotaxime, imipenem, and a narrow-spectrum cephem (cefazolin) was isolated from the urine of a patient treated with ß-lactam antibiotics. Resistance to ß-lactams was conjugatively transferred with a plasmid of about 50 kbp. The pI of this enzyme was 8.3. The sequence of the gene was determined, and the open reading frame of the gene was found to consist of 861 bases (GenBank accession number AY223863). Kinetic parameters showed that SHV-57 had a poor affinity to cefazolin. The K_m value toward cefazolin (5.57 x 10³ µM) was extremely high in comparison to those toward ceftazidime (30.9 µM) and penicillin G (67 µM), indicating its low affinity to cefazolin. Although the K_m value of the ß-lactamase inhibitor was too high for the study of catalytic activity (k_cat), indicating the low k_cat of SHV-57, the SHV-57 carrier was highly susceptible to a ß-lactam-ß-lactamase inhibitor combination. Comparison of the three-dimensional molecular model of SHV-57 with that of the SHV-1 ß-lactamase suggests that the substitution of arginine for leucine-169 in the loop is important for the substrate specificity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Since the first extended-spectrum ß-lactamase (ESBL) was isolated in Germany in 1983 (11), TEM-, SHV-, CTX-, and OXA-type ESBLs have been described in various members of the family Enterobacteriaceae (G. A. Jacoby and K. Bush, http://www.lahey.org/studies/webt.htm). Most of the ESBLs have altered hydrolytic activities compared with those of the classical enzymes TEM-1, TEM-2, and SHV-1 as a result of amino acid changes in different specific positions (10, 17). SHV-1 is a narrow-spectrum ß-lactamase with activity against penicillins. The first extended-spectrum SHV enzyme was described in 1985 and was named SHV-2 (10). The serine at amino acid position 238 was found to be replaced by glycine in SHV-1 and was found to cause resistance to extended-spectrum ß-lactams. Since then, many SHV-type ESBLs have been reported. Most of the substitutions are at Ambler position 179 or 238, alone or in combination with alterations at positions 35 and 240, which are important for substrate extension (G. A. Jacoby and K. Bush, http://www.lahey.org/studies/webt.htm). X-ray crystallography shows that mutations which cause amino acid changes on or close to the loop of the enzyme are highly correlated to resistance to extended-spectrum ß-lactams (12). The mutation at Gly238 has frequently been reported in SHV-type ESBLs. It causes resistance to various antibiotics, ranging from narrow-spectrum cephalosporins (cefazolin) to extended-spectrum cephalosporins (ceftazidime and cefotaxime). However, resistance only to extended-spectrum cephalosporins with susceptibility to narrow-spectrum cephalosporins is rarely encountered. In this report we delineate the mechanism of resistance of an Escherichia coli isolate recovered during an island-wide survey in Taiwan (7). This isolate is highly resistant only to ceftazidime but is susceptible to cefazolin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. E. coli 981223 was collected during an island-wide study of antibiotic resistance in Taiwan in 1998 (7). It was isolated in September 1998 from the urine of an 18-month-old boy with pneumonia. He had a history of failure to thrive, multiple abnormalities, intussusceptions, intestinal resection, and recurrent pneumonia that had resulted in 10 hospital admissions since birth. Before strain isolation, the patient had received empirical antibiotic treatment, including treatment with penicillin, oxacillin, cefotaxime, cefuroxime, ceftriaxone, amikacin, ceftazidime, vancomycin, erythromycin, and gentamicin, during the 10 hospitalizations. The strain was identified as E. coli with the Vitek system (bioMerieux Vitek, Inc., Hazelwood, Mo.).

Conjugation. The transfer of resistance was carried out by conjugation. A rifampin-resistant strain of E. coli (strain JP-995) (18) was used as the recipient. Recipients and donors were separately inoculated into brain heart infusion broth (Oxoid Ltd., Basingstoke, England) and incubated at 37°C for 4 h. They were then mixed at a ratio of 1:10 (by volume) for overnight incubation at 37°C. A 0.1-ml volume of the overnight broth mixture was then spread onto a MacConkey agar plate containing rifampin (100 µg/ml) and ceftazidime (2 µg/ml).

Susceptibility testing. Antimicrobial susceptibility was determined by the broth microdilution test, according to the guidelines of the National Committee for Clinical Laboratory Standards (16). The following antimicrobial agents were used: ampicillin, cephalothin, cefazolin, cefoxitin, cefotaxime, cefotaxime-clavulanic acid, ceftriaxone, ceftriaxone-clavulanic acid, ceftazidime, ceftazidime-clavulanic acid, imipenem, amikacin, aztreonam, and ciprofloxacin. All drugs except ciprofloxacin were incorporated into Mueller-Hinton broth (TREK Diagnostic System Ltd., Chichester, West Sussex, United Kingdom) in serial twofold concentrations from 0.25 to 32 µg/ml; a lower concentration of 0.03 µg/ml was used for ciprofloxacin. Two control strains, E. coli ATCC 35218 and ATCC 25922, were included in each test run. The inoculated plates were incubated at 35°C for 16 to 18 h. The MIC of each antimicrobial agent was defined as the lowest concentration that inhibited visible growth of the organism.

Isoelectric focusing. After 20 h of culture in brain heart infusion broth, the bacterial cells were harvested by centrifugation and the pellet was resuspended in 1 ml of phosphate buffer (10 mM; pH 7). Enzymes were released by two cycles of freezing at –70°C and thawing at room temperature and sonication for 5 min in a sonicator in ice-cold water. Isoelectric focusing was performed in an ampholine gel (pH 3.0 to 10.0; Pharmacia, Uppsala, Sweden). Preparations from standard strains known to harbor SHV-5 and CTX-M-14 were used as standards. After isoelectric focusing, ß-lactamases were detected by spreading nitrocefin (50 µg/ml) on the gel surface (14).

Cloning of SHV-57 gene. Plasmid DNA from the transconjugant was isolated with a plasmid mini kit (Qiagen, Inc., Mississauga, Ontario, Canada) and was partially digested with Sau3AI. The fragments were ligated into the BamHI site of pHSG298 by using T4 DNA ligase (Invitrogen, Carlsbad, Calif.) and electroporated into E. coli DH5. Clones were selected on Luria-Bertani agar plates containing 25 µg of kanamycin/ml and 5 µg of ceftazidime/ml.

DNA sequencing analysis. The plasmid containing the cloned bla_SHV-57 gene was prepared with a Concert Rapid Plasmid Miniprep system (GibcoBRL, Grand Island, N.Y.). The cloned gene was sequenced with the primers listed in Table 1. The N-terminal sequence was obtained by analyzing purified SHV-57 with a protein sequencer (PPSQ-23; Shimadzu, Kyoto, Japan) by the Edman degradation method (1). Mass spectrum analysis was done with an AXIMA-CFR plus mass spectrometer (Shimadzu).

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TABLE 1. Oligonucleotide primers used for amplification and/or sequencing reactions

Construction of overproduction system. XhoI and NdeI restriction sites were inserted into the plasmid DNA of the original SHV-57 enzyme. This was done during PCR amplification with the sense and antisense primers listed in Table 1. PCR amplification conditions were denaturation at 94°C for 5 min, followed by 25 cycles of amplification at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. The resulting PCR product was purified with a QIAquick PCR purification kit (Qiagen, Inc.). The amplified product was then inserted into plasmid vector pCR2 Blunt II TOPO (Invitrogen), and the plasmid was transformed into E. coli MV1184 [ara (lac-proAB) rpsL thi(80lacZM15) (srI-recA)306::Tn10(tet') F' traD36 proAB⁺ lacI^q lacZM15 (Takara Shuzo Co. Ltd., Shiga, Japan). Sequencing was done with an automatic sequencer (ABI Prism 310 genetic analyzer; Perkin-Elmer Biosystems, Norwalk, Conn.). After the sequence was confirmed, XhoI and NdeI (Takara Shuzo Co. Ltd., Tokyo, Japan) were used to digest the PCR product. The product was then cloned into the vector pET 28a (Novagen, Madison, Wis.).

Purification of ß-lactamase. E. coli BL21(DE3)pLysS, F^– ompT hsdS_B (r_B^– m_B^–) gal dcm (DE3)pLysS (Novagen), was used to produce the enzyme as a soluble protein. Since bacteria grown at 35 and 30°C produced inclusion bodies, a growth temperature of 25°C was used for the bacteria, which were incubated in 1 liter of Super broth on a rotating shaker. When the optical density at 600 nm of the culture reached an absorbance of 0.5, isopropyl-ß-D-thiogalactopyranoside (IPTG; Sigma, Steinheim, Germany) was added at concentrations of 0, 10, 50,100, 150, 200, 500, and 1 mM. A final concentration of 50 µM IPTG gave an appropriate level of enzyme expression. The maximum activity of the enzyme was reached 7 h after induction. The bacteria were incubated on ice for 10 min and then harvested by centrifugation at 5,000 x g for 10 min at 4°C. The cells were then suspended in 100 ml of 30 mM Tris-HCl buffer (pH 8.0) containing 27% sucrose. Liberation of the periplasmic content was achieved by addition of lysozyme (final concentration, 0.4 mg/ml) and EDTA (final concentration, 5 mM) to the cooled solution. After 50 min of incubation on ice, the reaction was stopped by adding CaCl₂ (final concentration, 2 mM). The sample was then centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was dialyzed overnight at 4°C in 5 liters of 10 mM sodium acetate buffer (pH 5.0). Purification was done with a HiPrep 16/10 SP XL system (Amersham Biosciences AB, Uppsala, Sweden) equilibrated with 10 mM sodium acetate buffer (pH 5.0). The initial rate of hydrolysis of 100 µM nitrocefin (₄₈₂ = +10,000 M^–1cm^–1; Oxoid Ltd.) was measured, and all fractions containing ß-lactamase activity were pooled. The purity of the ß-lactamase preparation was controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels stained with Coomassie brilliant blue. The purification process was done with an AKTA purifier (Amersham Biosciences AB).

Determination of kinetic parameters. The activity of the highly purified ß-lactamase was measured by spectrophotometric assay with a UV-2550 spectrophotometer (Shimadzu) connected to a personal computer. The rate of hydrolysis of the antibiotics by SHV-57 and SHV-1 was determined by monitoring the variation in the absorbance of each ß-lactam. Steady-state kinetic parameters were determined for the following ß-lactam compounds diluted in 50 mM phosphate buffer (pH 7.0). Benzylpenicillin (₂₃₃ = –780 M^–1cm^–1) was purchased from Sigma Chemical Co. (St. Louis, Mo.). Ceftazidime (₂₆₅ = –10,300 M^–1cm^–1) and clavulanic acid were gifts from Glaxo SmithKline (Tokyo, Japan). Cefazolin was a gift from Fujisawa Pharmaceutical Co. Ltd. (Osaka, Japan), and tazobactam was a gift from Taiho Pharmaceutical Co. (Tokyo, Japan). Four to seven different substrate concentrations were used to determine the kinetic parameters for each substrate, and the values for the parameters reported are averages of three independent measurements. The reactions were performed in a total volume of 500 µl at 30°C. Bovine serum albumin (20 mg/ml) was added to the enzyme to prevent denaturation. The values for all kinetic parameters were determined by measuring the initial rate of hydrolysis of the selected antibiotic and using Hanes-Wolf linearization of the Michaelis-Menten equation. In the case of poor substrates, the K_m was determined as the competitive inhibition constant (K_i) with nitrocefin as the reporter substrate.

Structural model of SHV-57. A structural model of SHV-57 was constructed by mutating Leu169 to arginine with the Biopolymer module installed in the Insight II program (version 2000; Accelrys Inc., San Diego, Calif.). The structure of the model was minimized with the Discover 3 program (version 2000; Accelrys Inc.) until the final root mean square deviation became less than 0.1 kcal/mol/Å. Ceftazidime and cefazolin were manually docked into the binding site of SHV-1 and SHV-57 by placing the carbonyl oxygen atom of the ß-lactam at the oxyanion hole formed by the amide groups of Ser70 and Ala237. The energies of the complex structures were minimized with the Discover 3 program, and the binding sites of the minimized structures were then covered with a sphere of water molecules of 20 Å in diameter centered at the Ser70 residue. The optimized complex structure was selected from 100 energy-minimized structures sampled by molecular dynamics calculations, which were performed at 300 K on residues within 12 Å from the ß-lactam compound with a cutoff distance of 10 Å, a distance-dependent dielectric constant, and a time step of 1 fs for 100 ps by sampling the conformation every 1 ps by using the Consistent Valence Force Field parameters in the Discover 3 program (version 98; Accelrys Inc.) at 298 K for 100 ps.

Nucleotide sequence accession number. The nucleotide sequence data for the SHV-57 gene were submitted to the National Center for Biotechnology Information Data Libraries (GenBank), and the sequence has been given accession number AY223863.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid profile and isoelectric focusing. The ß-lactam resistance of the isolate harboring the ß-lactamase was found to be conjugatively transferable. Only one plasmid was found in the recipient. The plasmids from the transconjugants had molecular sizes that ranged from approximately 40 to 60 kb. Transconjugants which acquired ceftazidime resistance by conjugation appeared at a frequency of 10^–5. The plasmid, which was designated pMTY512 (pMTY; registered with the Plasmid Reference Center), was cleaved into 16 segments by HincII. From the sizes of the fragments obtained, the size of pMTY512 was estimated to be about 51 kb. Narrow-range ampholine gel electrophoresis revealed that, with reference to the CTX-M-14 (pI 8.1) and SHV-5 (pI 8.2) ß-lactamases, the SHV-57 ß-lactamase had a pI of 8.3 (Fig. 1).

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FIG. 1. Isoelectric focusing of the new ß-lactamase variant and reference ß-lactamases. The numbers on the left are in kilobases.

Susceptibility testing. The SHV-57 carriers, transconjugants, and cloned strains were found to be ampicillin, cephalothin, and ceftazidime resistant. They were susceptible to cefazolin, cefotaxime, aztreonam, ciprofloxacin, amikacin, and imipenem. When clavulanic acid at a fixed concentration of 4 µg/ml was combined with ceftazidime, a greater than fourfold reduction in the ceftazidime MIC, a characteristic of ESBLs, was observed (Table 2).

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TABLE 2. MICs of various antibiotics for strains producing the SHV-57 ß-lactamase

DNA sequencing. The nucleotide sequence of 1,310 bp was determined by the strategy shown in Fig. 2. An 861-nucleotide open reading frame with a G+C content of 63.2% was present in this sequence. The sequence initiation codon (ATG) was preceded by a possible –10 region (AAAAAT) and a –35 region (TTGATT) of a putative promoter. The termination codon was TAA. From the putative open reading frame, the precursor form of SHV-57 seemed to consist of 286 amino acid residues. Consensus sequences, such as SXXK, SDN, and KTG, in class A ß-lactamases were found in the amino acid sequence of the SHV-57 ß-lactamase (GenBank accession number AY223863). Thus, SHV-57 is a class A ß-lactamase.

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FIG. 2. (A and B) Complex structure model of ceftazidime in SHV-57 and SHV-1, respectively. Ceftazidime is shown by the stick model. The carbon atoms are in light blue; and the oxygen, nitrogen, and sulfur atoms are in red, dark blue, and yellow, respectively, unless indicated otherwise. Only the main chain of SHV-57 within 12 Å of ceftazidime is shown by the ribbon model. Major residues proximal to ceftazidime are shown by the ball-and-stick model. The carbon atoms of the residues are in green, unless indicated otherwise. The residues are indicated by one-letter code. The red dotted lines indicate the hydrogen bonds between ceftazidime and the enzyme. (C and D) Complex structure models of cefazolin in SHV-57 and SHV-1, respectively. Cefazolin is shown by the stick model. Only the main chain of SHV-57 within 12 Å of cefazolin is shown by the ribbon model. Major residues proximal to cefazolin are shown by the ball-and-stick model. The colors of the various elements are as defined for panels A and B.

Determination of kinetic parameters. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 28,904. The overproduction system and purification process yielded 1.6 mg of purified SHV-57 per ml in a total volume of 2.5 ml. The purity achieved was over 95%, as observed by SDS-PAGE. The N-terminal sequence of the mature enzyme is SPQPLEQIKLSESQLSGRVGMIEMDLASGRTLTAWRADERFPMMSTFK. The kinetic parameters for SHV-57 and SHV-1 are summarized in Table 3. The results showed that the SHV-57 ß-lactamase exhibited a narrow-spectrum activity profile, although notable differences were detected with different substrates. The K_m value toward cefazolin (5.57 x 10³ µM) was extremely high compared to those toward ceftazidime (30.9 µM) and penicillin G (67 µM), indicating its low affinity to cefazolin. The K_i values of clavulanic acid and tazobactam for the inhibitors were 27 x 103 and 1.16 x 10³ µM, respectively. Similarly, the concentration of cefazolin required for the study of catalytic activity (k_cat) was also too high and could not be detected with our equipment, indicating the low catalytic activity of SHV-57. On the other hand, SHV-57 had relatively higher catalytic activities for ceftazidime and penicillin G than for cefazolin. The hydrolytic efficiencies (k_cat/K_m) of the purified enzyme toward benzylpenicillin and ceftazidime were 5.67 x 10^–5 and 2.78 x 10^–5 µM^–1 s^–1, respectively. Since the hydrolytic efficiency (k_cat/K_m) of cefazolin was not detectable, ceftazidime and penicillin G are relatively good substrates for SHV-57.

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TABLE 3. Comparison of kinetic parameters between SHV-57 and SHV-1 ß-lactamases

Structural model of SHV-57. The substitution of Arg for Leu169 induced a conformational change in the Asn170 residue, which was located at the site proximal to the moieties attached to the C-7 position of the cephalosporin skeleton. Thus, the aminothiazole moiety of ceftazidime bound favorably in the pocket formed by Asn170 and Glu240, which were located within hydrogen bond distances of the positively charged amino group of the aminothiazole moiety. In addition, Asn170 formed a hydrogen bond with the carboxylate group of the dimethyl-carboxymethyloxime moiety (Fig. 2A). Since in the crystal structure of SHV-1 Asn170 has a different conformation which does not permit the formation of favorable hydrogen bonds with ceftazidime (Fig. 2B), the hydrogen bonds should play a crucial role in the binding of ceftazidime in SHV-57. Thus, ceftazidime is a good substrate for SHV-57. In contrast, the tetrazole moiety of cefazolin is electrostatically negative, and thus, interactions between the tetrazole moiety and Asn170 are unfavorable. Moreover, an unfavorable electrostatic interaction between negatively charged Glu240 and the tetrazole moiety dislocated the tetrazole moiety away from the binding site (Fig. 2C). Asn170 in SHV-1 does not interfere with the binding of the tetrazole moiety, which formed a favorable hydrogen bond with Thr167. In addition, the tetrazole moiety had a conformation that avoided an unfavorable electrostatic interaction with the carboxylate group of Glu240. Therefore, whereas SHV-1 binds to cefazolin with favorable interactions at the tetrazole binding site (Fig. 2D), SHV-57 has unfavorable interactions with the tetrazole moiety; and thus, cefazolin is stable against SHV-57.

Top Abstract Introduction Materials and Methods Results Discussion References

 
SHV-57 is a plasmid-encoded class A ESBL. Most SHV-type ESBLs have the Gly238Ser substitution alone or combined with alterations at position 240 or 35 (G. A. Jacoby and K. Bush, http://www.lahey.org/studies/webt.htm). The substitution at amino acid position G238S is on the ß strand (13). It is the premier substitution that preserves penicillin and cephalosporin resistance (8). Another common group of SHV-type ESBLs has a single amino acid substitution at the Asn179 residue on the loop. These include SHV-6 (D179A), SHV-8 (D179N), and SHV-24 (D179G), which confer resistance to ceftazidime (12). The role of Asp179 has been well studied (2, 3, 12). The X-ray crystal structure of the mutant with the D179N mutation of the PC1 ß-lactamase shows a disordered loop (5). Crystallographic investigations of the structures of several class A ß-lactamases have shown a salt bridge between Arg164 and Asp179 that anchors the base of the loop (4-6, 9, 12, 19). Vakulenko et al. (20) changed Asp179 to 19 other amino acids by site-directed mutagenesis to disrupt the salt bridge between Arg164 and Asp179. Most of the substitutions for Asp179 increased the level of resistance to ceftazidime.

In our study, MIC data indicated that in SHV ESBLs, in addition to residue 179, residue 169 on the loop also plays an important role in influencing substrates. Interestingly, the susceptibility pattern showed that the enzyme caused resistance to ceftazidime but susceptibility to cefazolin and is inhibited by the ß-lactamase inhibitor clavulanic acid. On the contrary, kinetic studies showed that the ß-lactamase inhibitors tazobactam and clavulanic acid did not have inhibitory actions on this enzyme. A discrepancy between MIC and kinetic data has also been described for other ß-lactamases, such as MOX-1 (1). Perhaps the results of experiments conducted with pure enzyme in vitro are different from those conducted in vivo, in that bacteria can have other biochemical modifications under normal conditions. This contradiction between MIC and kinetic data needs further study.

SHV-57 is a clinical variant found in Taiwan. Because of the significant role that amino acid substitution plays in ß-lactamase-mediated resistance, we elucidated the substrate recognition mechanism using a model for the enzyme-substrate complex. In conclusion, the substitution of arginine for leucine-169 in the loop is important for substrate specificity and causes ceftazidime resistance.

 
This work was supported by the National Health Research Institutes of Taiwan.

 
* Corresponding author. Mailing address: Division of Clinical Research, National Health Research Institutes (99), 128, Yen-Chiu-Yuan Rd., Sec. 2., Taipei, 11529, Taiwan. Phone: 886 2 26524094. Fax: 886 2 27890254. E-mail: lksiu{at}nhri.org.tw.

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Antimicrobial Agents and Chemotherapy, February 2005, p. 600-605, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.600-605.2005
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