Characterization of a laboratory-generated variant of BPS ß-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime

P. L. Ho1,*, Terence K. M. Cheung1, W. C. Yam1 and K. Y. Yuen2

1 Division of Infectious Diseases, Department of Microbiology and 2 HKU-Pasteur Research Centre, Faculty of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Pokfulam, Hong Kong SAR, China

Received 11 April 2002; returned 3 July 2002; revised 14 August 2002; accepted 20 August 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Burkholderia pseudomallei produces an Ambler class A ß-lactamase, known as BPS-1. The ß-lactamase gene from a laboratory-derived, ceftazidime-resistant strain of B. pseudomallei (LH-1-2) was cloned and expressed in Escherichia coli. The ß-lactamase, named BPS-1m, had an identical isoelectric focusing point (pI 7.7) to that of BPS-1, but differed in having a stronger hydrolytic activity against ceftazidime. Susceptibility testing showed that BPS-1m when expressed in E. coli conferred resistance to ceftazidime (MIC >= 32 mg/L). The amino acid sequence of BPS-1m differed from that of BPS-1 by a Pro-to-Ser change at position 167 in the omega loop.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Burkholderia pseudomallei is intrinsically resistant to ampicillin and cefuroxime through the production of chromosomal ß-lactamases. One ß-lactamase, named BPS-1, was recently cloned and sequenced.1 BPS-1 contains the four conserved motifs, 70SXXK73, 130SDN132, 166EXXXN170 and 234KTG236 (Ambler’s numbering scheme), that are characteristic of class A ß-lactamases. The enzyme hydrolysed some cephalosporins, such as cephalothin and cefuroxime, strongly, but ceftazidime was not inactivated. Emergence of a mutant ß-lactamase that caused resistance to ceftazidime has been described during treatment of melioidosis with the antibiotic, but no genetic characterization of the clinical isolates was carried out.2 Here, we report on the cloning and expression of a ß-lactamase from a laboratory-derived strain of ceftazidime-resistant B. pseudomallei.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains

B. pseudomallei strain LH-1 was isolated in 1999 in Hong Kong from a blood sample of an adult patient with pulmonary melioidosis. The isolate was identified by Gram’s stain, colonial morphology on Ashdown medium, oxidase reaction, resistance to polymyxin B, the API 20NE system (BioMérieux SA, France) and a specific agglutination kit (kindly provided by V. Wuthiekanan, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand).3

Susceptibility testing

The MICs of ampicillin, co-amoxiclav, cephaloridine, cephalothin, cefuroxime, ceftazidime, cefepime, cefpirome, imipenem and meropenem were determined by Etest (AB Biodisk, Solna, Sweden) or an agar dilution method, and interpreted according to NCCLS criteria.4 Control strains Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were included with each run.

Selection of a ceftazidime-resistant mutant from LH-1

B. pseudomallei strain LH-1 (ceftazidime MIC 4 mg/L) was induced to develop resistance to ceftazidime by daily subculture in Luria–Bertani (LB) broth containing an increasing concentration of ceftazidime (2, 4, 8, 12, 16, 20, 24, 32, 48 and 64 mg/L). At the end of the ninth passage, a resistant mutant (LH-1-2) with a ceftazidime MIC >= 32 mg/L was obtained. Resistant cells in the final broth were subcultured and four separate colonies were picked for sequence analysis.

Cloning experiments, analysis of recombinant plasmid and sequencing

Cloning of the ß-lactamase gene from the ceftazidime-resistant B. pseudomallei mutants (LH-1-2) was performed by a PCR cloning strategy as described previously.1 Plasmid pBK-CMV and E. coli XL1-Blue MRF' (Stratagene, La Jolla, CA, USA) were used as the cloning vector and recipient strain, respectively. Clones were selected on LB agar supplemented with ceftazidime (24 mg/L) and kanamycin (50 mg/L). Sequencing of the BPS PCR products from the wild-type strain, the laboratory mutants and the recombinant plasmids was performed by cycle sequencing. The primers F2 forward (position –9 to 11) and B2 backward (position 894–875), which amplified the whole BPS gene, were used for both PCR and sequencing.1 The sequences of both strands of the PCR products were determined by the Bigdye dideoxynucleotide chain termination method with an ABI PRISM 377 Genetic Analyser (Perkin-Elmer Corp., Foster City, CA, USA).

ß-Lactamase assays and isoelectric focusing analysis

ß-Lactamase was extracted by sonication as described previously.1 Membrane-bound ß-lactamase was released by a single extraction with Triton X-100. Isoelectric focusing of the ß-lactamases was performed with ampholine gel (Pharmacia, Hong Kong, China) from pI 3.5 to pI 9.5. Enzyme extracts from strains expressing TEM-1, OXA-1 and SHV-1 were used as controls. The pI value of each enzyme was determined by overlaying the gel with nitrocefin. Crude lysate without purification was used for substrate and inhibition assays, as described previously.1 Antibiotic powders with known potencies were provided by the manufacturers (cefpirome, clavulanate and sulbactam) or purchased from Sigma (St Louis, MO, USA).

Nucleotide sequence accession number

The sequence of blaABPS–1m has been given GenBank accession number AY082515.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Isoelectric focusing analysis of ß-lactamases extracted from the parent strain (LH-1), the mutant (LH-1-2) and an E. coli clone (P-LH-1-2, containing plasmid pBK-CMV-BPS-1m) revealed an enzyme with the same isoelectric point (pI) of 7.7. This was the only ß-lactamase in P-LH-1-2, but there were two more bands at pI 8.0 and 8.2 for the extracts from LH-1 and LH-1-2. The ß-lactam susceptibility profiles of these strains are shown in Table 1. The kinetic parameters of the ß-lactamase with pI 7.7 obtained from E. coli clone P-LH-1-2 and the crude extracts from LH-1 and LH-1-2 are shown in Table 2.


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Table 1. MICs of ß-lactams for reference and ß-lactamase (BPS-1 or BPS-1m)-producing strains
 

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Table 2. Kinetic parametersa of BPS-1 and BPS-1m ß-lactamases
 
The sequence of the PCR-amplified ß-lactamase gene from clinical strain LH-1 was identical to blaABPS-1 described by our group recently.1 Analysis of the PCR-amplified ß-lactamase gene from LH-1-2 and the DNA insert cloned in pBK-CMV-BPS-1m both showed an identical 888 nucleotide open reading frame that encoded a 295 amino acid polypeptide. The sequence of the ß-lactamase gene (named blaABPS-1m) differed from blaABPS-1 by one base at nucleotide position 517 (CCT->TCT), leading to Pro167 (Ambler numbering) being substituted by Ser.

The MICs of ß-lactams showed that BPS-1m, when expressed in E. coli XL1-Blue MRF' (pBK-CMV-BPS-1m), conferred resistance to ampicillin and ceftazidime. The E. coli clone remained susceptible to co-amoxiclav, indicating that the mechanism of resistance was conferred by ß-lactamase. Kinetic properties of BPS-1m (E. coli P-LH-1–2) differed from BPS-1 (E. coli P1) in several aspects. First, it had a higher ceftazidime hydrolytic activity. Secondly, BPS-1m had higher affinity for cephaloridine and cefuroxime, but hydrolysed them at a relative lower rate. Our findings thus agreed with the characteristics of the ß-lactamase produced by a ceftazidime-resistant clinical isolate of B. pseudomallei.2

The Triton-treated extract from the two parent strains gave three bands in the isoelectric focusing, whereas the E. coli clone only gave a single band at pI 7.7. These bands could correspond to other ß-lactamases described recently.5 Alternatively, the bands at pI 8.0 and 8.2 could represent membrane-bound derivatives from the same ß-lactamase gene. This phenomenon has been described for the membrane-bound ß-lactamases BRO-1 and BRO-2.6

In this study, a single amino acid substitution (Pro167->Ser) in the omega loop was found to increase the ceftazidime hydrolytic activity of the ß-lactamase. Among the Ambler class A enzymes, it is known that residues in the omega loop play an important role in the substrate profile for cephalosporins.7 In TEM-1, amino acid substitutions in the omega loop (residues 162–179) were found to increase the level of activity towards ceftazidime.8 Substitutions that expand the catalytic activity typically involve replacement of a larger amino acid, such as proline, by a small amino acid, such as serine or glycine.8 In terms of amino acid homology, BPS ß-lactamase has a high degree of similarity to the CTX-M ß-lactamases that are prevalent in South America.1 In agreement with our finding, an identical amino acid substitution at residue 167 (Pro->Ser) was found to convert CTX-M-18 into a ceftazidime-hydrolysing ß-lactamase.9

In conclusion, the sequence of a BPS ß-lactamase that hydrolyses ceftazidime is reported. This enzyme, BPS-1m, differs from the wild-type ß-lactamase by a single amino acid substitution at residue 167 (Pro->Ser).


    Acknowledgements
 
This work was supported by a grant from the Research Grant Council (HKU 7282/97 M), Hong Kong Special Administrative Region, China.


    Footnotes
 
* Corresponding author. Tel: +852-2855-4897; Fax: +852–2855-1241; E-mail: plho{at}hkucc.hku.hk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cheung, T. K., Ho, P. L., Woo, P. C., Yuen, K. Y. & Chau, P. Y. (2002). Cloning and expression of class A ß-lactamase gene blaABPS in Burkholderia pseudomallei. Antimicrobial Agents and Chemotherapy 46, 1132–5.[Abstract/Free Full Text]

2 . Godfrey, A. J., Wong, S., Dance, D. A., Chaowagul, W. & Bryan, L. E. (1991). Pseudomonas pseudomallei resistance to ß-lactam antibiotics due to alterations in the chromosomally encoded ß-lactamase. Antimicrobial Agents and Chemotherapy 35, 1635–40.[ISI][Medline]

3 . Smith, M. D., Wuthiekanun, V., Walsh, A. L. & Pitt, T. L. (1993). Latex agglutination test for identification of Pseudomonas pseudomallei. Journal of Clinical Pathology 46, 374–5.[Abstract]

4 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement, Vol. M100-S10. NCCLS, Wayne, PA, USA.

5 . Hartman, G. C., Keith, K. E., Crossett, B., Titball, R. W., Brown, K. A. & Walsh, T. R. (2001). Identification and characterization of a class A and class C ß-lactamase and their AmpR regulators in Burkholderia pseudomallei. In Program and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract 583. American Society for Microbiology, Washington, DC, USA

6 . Eliasson, I., Kamme, C., Vang, M. & Waley, S. G. (1992). Characterization of cell-bound papain-soluble beta-lactamases in BRO-1 and BRO-2 producing strains of Moraxella (Branhamella) catarrhalis and Moraxella nonliquefaciens. European Journal of Clinical Microbiology and Infectious Diseases 11, 313–21.[ISI][Medline]

7 . Banerjee, S., Pieper, U., Kapadia, G., Pannell, L. K. & Herzberg, O. (1998). Role of the omega-loop in the activity, substrate specificity, and structure of class A beta-lactamase. Biochemistry 37, 3286–96.[ISI][Medline]

8 . Palzkill, T., Le, Q. Q., Venkatachalam, K. V., LaRocco, M. & Ocera, H. (1994). Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase. Molecular Microbiology 12, 217–29.[ISI][Medline]

9 . Poirel, L., Naas, T., Le Thomas, I., Karim, A., Bingen, E. & Nordmann, P. (2001). CTX-M-type extended-spectrum beta-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrobial Agents and Chemotherapy 45, 3355–61.[Abstract/Free Full Text]