Cloning of the class D ß-lactamase gene from Burkholderia pseudomallei and studies on its expression in ceftazidime-susceptible and -resistant strains

Pannika Niumsup1,* and Vanaporn Wuthiekanun2

1 Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanuloke 65000; 2 Wellcome Trust Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

Received 10 January 2002; returned 15 April 2002; revised 11 June 2002; accepted 3 July 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ceftazidime is the antibiotic of choice for the treatment of melioidosis. Ceftazidime-resistant Burkholderia pseudomallei have been identified and ß-lactamase production implicated in resistance. In this study, 25 strains of B. pseudomallei (15 clinical and 10 environmental strains) were examined for their ability to yield mutants that overexpress ß-lactamase. Ceftazidime-resistant mutants were selected readily at high frequency and displayed four- to eight-fold increases in the MICs of ceftazidime. ß-Lactamase activities in both parent and mutant B. pseudomallei strains were examined by a spectrophotometric method. Twelve mutants (48%) showed approximately two- to 31-fold higher ceftazidimase activity compared with their parent strains and 10 (40%) demonstrated more than two-fold increases in imipenemase activity. A class D ß-lactamase gene from B. pseudomallei was cloned and sequenced. The encoded enzyme is an oxacillinase and is homologous to oxacillinases from Ralstonia pickettii and members of the genus Aeromonas. Reverse transcriptase PCR showed that transcription of the class D ß-lactamase gene is increased in ceftazidime-resistant mutants.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Melioidosis, a fatal disease caused by Burkholderia pseudomallei, is endemic in Thailand, especially in the north-east. Patients with systemic B. pseudomallei infection require prompt antimicrobial therapy, as the mortality rate is exceptionally high.1 Currently, the antibiotic of choice for the treatment of acute melioidosis is ceftazidime.2 Resistance to ceftazidime was recognized soon after this antibiotic became the treatment of choice for severe melioidosis. Resistance was shown to be the result of ß-lactamase production.3,4 The carbapenems were therefore evaluated as an alternative treatment, although their use is still limited.5 Furthermore, in some other Gram-negative pathogens such as Enterobacter cloacae6 and Acinetobacter baumannii,7,8 carbapenem resistance has emerged, particularly because of the production of specific ß-lactamases.

To date, there is relatively little information on ß-lactamase expression in B. pseudomallei. In 1987, Livermore et al.9 reported that nine strains of B. pseudomallei possess a weakly inducible cephalosporinase that is active against carbenicillin, cefotaxime and cefuroxime. Later, Dance et al.3,4 noted the presence of a clavulanic acid-susceptible ceftazidimase. Godfrey et al.10 reported that ß-lactam resistance in B. pseudomallei resulted from overexpression of chromosomal ß-lactamases, and this has also been shown to result in high-level resistance to ß-lactams in Burkholderia cepacia.11 In contrast, 194 strains of B. pseudomallei were examined for ß-lactamase expression by Sookpranee et al.,12 who concluded that the ß-lactamases found were non-inducible.

Carbapenemases have recently become more prominent among ß-lactam-hydrolysing enzymes, and have also been described in carbapenem-resistant B. cepacia.13 This organism produces an inducible metallo-ß-lactamase, designated PCM-1, that hydrolyses imipenem, meropenem and, to a lesser extent, ceftazidime. Therefore, it is not unreasonable to speculate that carbapenem-hydrolysing enzymes may also be present in B. pseudomallei. However, sequencing of the genome of B. pseudomallei strain K96243 has revealed the presence of class A, C and D ß-lactamase genes, but not a class B (metallo-)ß-lactamase gene (Sanger Institute, Cambridge, UK). The phenotypic significance of the ß-lactamase genes located has not yet been elucidated.

When bacteria carrying a ß-lactamase gene(s) are exposed to ß-lactams, they come under strong selective pressure to develop resistance. Many Gram-negative bacteria, including B. cepacia, have been reported to produce, at high frequencies, mutants that overexpress ß-lactamase.11 In this study, environmental and clinical strains of B. pseudomallei were examined for their abilities to yield mutants that overexpress ß-lactamase expression. The frequency of mutation to ceftazidime resistance, and the ß-lactam resistance profiles and levels of ß-lactamase production of the resultant mutants, were investigated. The genes encoding class D ß-lactamases were cloned and sequenced from two B. pseudomallei strains. Expression of this gene in one ceftazidime-resistant mutant compared with its parent was also measured.


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

The clinical B. pseudomallei strains (1901a–1928a) used in this study were isolated from patients in Ubon Ratchatani, north-east Thailand. Environmental strains (E10–E188) were isolated from rice fields in the north-east of Thailand (Table 1).


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Table 1.  Time and area of isolation of B. pseudomallei strains used in this study
 
Selection of ceftazidime-resistant mutants

Ceftazidime-resistant mutants were selected by plating an overnight culture of B. pseudomallei onto ceftazidime-containing Mueller–Hinton agar at 4 x MIC. The same culture was also diluted, plated on agar without antibiotic and incubated at 37°C to determine the viable cell count. The mutation frequency was calculated as the number of resistant colonies compared with the number of cells in the original culture.

Susceptibility testing

MICs were determined by a standard agar dilution method. Inocula were prepared by diluting an overnight culture of each strain. Using a multipoint inoculator, ~104 cells were spotted onto dried Mueller–Hinton agar containing serial dilutions of the appropriate antibiotic. Antibiotic concentrations ranging from 0.125 to 512 mg/L with doubling increments were used. After 18 h incubation at 37°C, the MIC was recorded as the lowest concentration of antibiotic that inhibited visible growth.

Preparation of crude cell extracts containing ß-lactamase

The B. pseudomallei cultures were grown until mid-log phase. Then, toluene was added to the cultures in order to kill bacteria, as described by Livermore et al.9 The cells were harvested and washed once with 10 mM phosphate buffer (pH 7.0). The cultures were then disrupted on ice by four cycles of 20 s sonications with 15 s rest intervals between cycles (Vibra Cell; Sonics & Materials Inc., Newtown, CT, USA), and centrifuged at 13 000 rpm at 4°C for 10 min (Beckman J2-MC; Beckman, CA, USA). The supernatant was used in ß-lactamase assays.

Crude cell extracts from Escherichia coli carrying the oxacillinase gene were prepared by periplasmic extraction as described by Lindström et al.14

ß-Lactamase assays

ß-Lactamase activities were measured by monitoring hydrolysis of ß-lactams by spectrophotometric assay (Spectronic GenesysTM 5; Milton Roy Company, Rochester, NY, USA) at a wavelength of optimal absorbance for the ß-lactam ring of each drug.9,15 Antibiotic solutions were prepared freshly in 10 mM phosphate buffer (pH 7.0). Ceftazidime, imipenem (Merck & Co., Inc., West Point, PA, USA), ampicillin and cefalothin (Sigma, St Louis, MO, USA) were assayed at concentrations of 100, 300, 500 and 100 µM, respectively. Prior to each assay, a background reading of each antibiotic was performed to ensure that any decrease in substrate was solely the result of adding the enzyme extracts. All assays were run in triplicate. The protein concentration was measured using a Bio-Rad protein assay based on the Lowry method (Bio-Rad, Hercules, CA, USA). Specific activities were calculated as nanomoles of ß-lactam hydrolysed per minute per milligram of protein.

Amplification and cloning of class D ß-lactamase genes from B. pseudomallei

Chromosomal DNA was isolated from B. pseudomallei parent strains 1902a and E15 using an Easy-DNA kit (Invitrogen, Groningen, The Netherlands), and was used directly as a template in PCR. The primers used were based on the genome sequence of B. pseudomallei K96243, which is available online (at http://www.sanger.ac.uk/Projects/B_pseudomallei/). PCR primers were purchased from Sigma-Genosys Ltd (Pampisford, UK). The class D gene primers were: (forward) 5'-ATGAGCCATTCCCCACTCTT-3' and (reverse) 5'-TTTTGCCGTTCACGAAGAC-3', which are 32 bp upstream from the start codon and 83 bp downstream from the stop codon, respectively. The predicted PCR product was ~900 bp. Reactions were performed with 50 µL of mixture containing 100 ng of template, 0.25 µM each oligonucleotide primer, 200 µM dNTPs and 1 U of SuperTaq (HT Biotechnology Ltd, Cambridge, UK) together with its reaction buffer. The conditions comprised one cycle at 94°C for 5 min, followed by 50 cycles at 94°C for 1 min, 55°C for 1.3 min and 72°C for 2.3 min, and a final elongation at 72°C for 10 min.

The amplified class D gene products generated by PCR were cloned into the pCR T7/CT-TOPO vector (Invitrogen), according to the manufacturer’s instructions. The recombinant plasmids were transformed into chemically competent E. coli TOP10F' cells by heat shock, as described by the supplier. Transformants were plated on nutrient agar containing ampicillin (100 mg/L) and zeocin (50 mg/L), and incubated at 37°C overnight. Ten colonies were picked randomly and recombinant plasmids were isolated using the Hybaid Recovery Plasmid Prep Kit (Hybaid, Teddington, UK). The orientation of the inserts was verified by PCR.

Measuring expression of class D ß-lactamase genes in B. pseudomallei using reverse transcriptase–PCR (RT–PCR)

Total RNA from B. pseudomallei, both parent and mutant strains, was isolated by Hybaid RiboLyser Kit BLUE using a HYBAID RiboLyser Instrument. The RNA was quantified and cDNAs were synthesized using RevertAid H Minus M-MuLV Reverse Transcriptase (MBI Fermentas, Vilnius, Lithuania), according to the supplier’s instructions. RNAs (5 µg) were mixed with specific primers, incubated at 70°C for 5 min and chilled on ice. Then, reaction buffer was added, followed by dNTPs to a final concentration of 1 mM. This reaction was incubated at 70°C for 5 min. Two hundred units of RevertAid H Minus M-MuLV Reverse Transcriptase were added and incubated at 42°C for 60 min. Finally, the reaction was terminated by heating at 70°C for 10 min and chilled on ice. The synthesized cDNA was used directly as template in RT–PCR without further purification. The oligonucleotide primers were designed within the gene: (forward) 5'-GCTGCTGGTGCAGGACGGCG-3' and (reverse) 5'-CGTCATGTCGACGGCTGTG-3'. The predicted PCR product was 435 bp. Amplification reactions were performed as described earlier and the PCR conditions were as followed: one cycle at 94°C for 5 min, followed by 50 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 2 min, with a final elongation at 72°C for 10 min. The amplified products were analysed on 1% agarose gel electrophoresis.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation of ceftazidime-resistant B. pseudomallei mutants

Ceftazidime-resistant mutants of 25 B. pseudomallei strains (15 clinical and 10 environmental strains) were selected by plating overnight cultures of B. pseudomallei onto ceftazidime-containing agar. It should be noted that the ‘ceftazidime-resistant mutant’ in this article refers to strains having four- to eight-fold increases in the MIC of ceftazidime compared with the parent strain. The mutation frequencies determined are shown in Table 2.


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Table 2.  Frequencies of mutation and ß-lactam susceptibilities of B. pseudomallei strains used in this study
 
Ceftazidime-resistant mutants from clinical isolates were selected at frequencies of 10–6–10–9 (mostly 10–8). For environmental isolates, the mutation frequencies (for selecting ceftazidime-resistant mutants) were slightly lower (10–9–10–10). The frequencies of mutant isolation suggested that point mutations leading to gene disruption were most likely to be responsible for the resistant phenotype, as seen in B. cepacia,11 Aeromonas spp.16 and Stenotrophomonas maltophilia.17 One mutant of each strain was chosen randomly for further investigation.

ß-Lactam susceptibilities and ß-lactamase production of B. pseudomallei, parent and mutant strains

The MICs of ceftazidime and imipenem against 25 parent and ceftazidime-resistant B. pseudomallei mutants were determined. The ranges of MICs against all strains are shown in Table 2. The parent strains were susceptible to both ceftazidime and imipenem. The mutants displayed up to eight- and four-fold increases in MICs of ceftazidime and imipenem, respectively.

To investigate whether the increase in MICs of ceftazidime and imipenem was the result of ß-lactamase production, ß-lactamase activities were determined, specifically against imipenem and ceftazidime. The imipenemase and ceftazidimase activities of 25 B. pseudomallei strains (both parent and mutant strains) are shown in Table 3. Twelve ceftazidime-resistant mutants (48%) (1901a, 1902a, 1912a, 1921a, 1922a, 1924a, 1926a, 1928a, E15, E23, E37, E187) showed two- to 31-fold increases in activities against ceftazidime. Increased imipenemase activity was found in 10 ceftazidime-resistant mutants (40%), which displayed two- to 52-fold higher activities compared with their parent strains (1901a, 1902a, 1907a, 1921a, 1922a, 1924a, 1928a, E25, E37, E187). Ceftazidime-resistant mutants of strains 1901a, 1902a, 1921a, 1922a, 1924a, 1928a, E37 and E187 (31% of all mutants) displayed a more than two-fold increase in activity against both imipenem and ceftazidime. Ceftazidime-resistant mutants of strains 1904a, 1911a, 1914b, 1916a, 1918a, 1923a, E10, E14, E19, E26 and E188, despite increases in MICs, displayed ß-lactamase activities similar to their parents. Ceftazidime resistance in these mutants may have resulted from other mechanisms, e.g. alteration in outer membrane proteins or drug efflux.


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Table 3.  ß-Lactamase activities of B. pseudomallei parent strains and mutant progeny
 
Amplification, sequencing and cloning of class D ß-lactamase genes from B. pseudomallei

The genome sequence of B. pseudomallei has revealed the presence of class A (contig 93), class C (contig 421) and class D (contig 14) ß-lactamase genes. B. pseudomallei strains 1902a and E15 were randomly chosen as representatives of clinical and environmental strains, respectively, for cloning of the class D ß-lactamase genes. Genomic DNAs were extracted and amplification of class D ß-lactamase genes from B. pseudomallei, strains 1902a and E15, were performed. A single, discrete band of ~900 bp was obtained for each strain, which was consistent with the predicted PCR product for a class D ß-lactamase gene.

Sequencing of the PCR product from strain 1902a revealed an open reading frame of 810 nucleotides, encoding a product of 269 amino acids. The nucleotide sequence of this open reading frame and its deduced amino acids are shown in Figure 1 (EMBL accession number AJ488302). The GC content is high throughout the open reading frame (65.9%), which corresponds to the overall GC content of the B. pseudomallei chromosome. This new class D ß-lactamase, designated OXA-42, has a predicted pI of 9.4 and an estimated molecular weight of 29.5 kDa. Like other ß-lactamases, the primary translation product has a strong hydrophobic N-terminus, typical of periplasmic proteins. The predicted cleavage site is likely to be between alanine and lysine at position 23–24. Within the enzyme, the conserved Ser-Thr-Phe-Lys active site was found at position 53–56. Four other conserved regions for class D ß-lactamases were also identified (Figure 1): Tyr-Gly-Asn (position 130–132); Trp-Xaa-Gly-Xaa-Xaa-Leu-Xaa-Ile-Ser (position 149–157), Gln-Xaa-Xaa-Xaa-Leu (position 161–165); and Lys-Thr-Gly (position 201–203).



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Figure 1. Nucleotide and deduced amino acid sequences of class D ß-lactamase from B. pseudomallei strain 1902a. Bold letters represent the conserved region of class D ß-lactamase. The putative ribosome binding site is underlined.

 
The predicted amino acid sequence of OXA-42 (from strain 1902a) is almost identical to the gene sequenced by the Sanger Institute (from strain K96243) with one amino acid difference. When OXA-42 was compared with other ß-lactamases in the EMBL database, this protein displayed high homology with several oxacillin-hydrolysing ß-lactamases such as OXA-22 from Ralstonia pickettii18 (51% identity); AsbB1, AmpS and AmpH, the class D enzymes from Aeromonas jandaei,19 Aeromonas veronii20 and Aeromonas hydrophila,15 respectively (45% identities); OXA-18 from Pseudomonas aeruginosa21 (43% identity); and OXA-9 from Klebsiella pneumoniae encoded on multiresistant transposon Tn131122 (41% identity).

The nucleotide sequence of the amplified class D ß-lactamase gene from B. pseudomallei strain E15 was also determined (EMBL accession number AJ488303). The deduced amino acid sequence, designated OXA-43, is virtually identical to that of OXA-42, differing by two amino acids. Figure 2 presents the alignment of OXA-42 and OXA-43 with the deduced amino acid sequence of the class D ß-lactamase gene from B. pseudomallei strain K96243, OXA-22, AsbB1, AmpH, OXA-18 and OXA-9.



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Figure 2. Alignment of class D ß-lactamase from B. pseudomallei 1902a and E15, OXA-42 and OXA-43, respectively, with the deduced amino acid sequence of the class D gene from B. pseudomallei strain K96243 and other related oxacillinases, such as OXA-22,17 OXA-18,20 OXA-9,21 AsbB118 and AmpH.15 The two amino acid differences between OXA-42 and OXA-43 are underlined and indicated by ‘{downarrow}’. Amino acid sequences that are found in all eight class D ß-lactamases are indicated by an asterisk. ‘:’ indicates an amino acid that is found in at least five proteins.

 
Prevalence of class D ß-lactamase genes in B. pseudomallei

Eleven B. pseudomallei strains were investigated for the presence of class D ß-lactamase genes. These strains were isolated from Thailand (1901a, 1921a), Vietnam (52.239, 56.91, A203, 52.238, 59.6, A202, 52.237, 55.135) and Australia (60.66) (Table 1). Chromosomal DNAs were isolated and used as templates in PCR amplification. The oligonucleotide primers and PCR conditions were exactly the same as those for strains E15 and 1902a. Strains 1902a and E15 were also included in this amplification as positive controls. The PCR products obtained from eight strains (except 60.66, 52.238 and 52.237) were ~900 bp in length, which corresponded to the predicted PCR product size, regardless of the geographical origin and isolation time (Figure 3 and Table 1). The PCR products were sequenced at both ends to verify that these PCR products were the class D ß-lactamase genes (data not shown).



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Figure 3. One percent agarose gel electrophoresis of amplification of class D ß-lactamase from B. pseudomallei. Lane 1, 1 kb plus DNA ladder; lanes 2–14, class D PCR products from B. pseudomallei strains 52.239, 56.91, 60.66, A203, 52.238, 59.6, A202, 52.237, 55.135, 1901a, 1902a, 1921a and E15, respectively.

 
ß-Lactam susceptibilities and ß-lactamase activities of the E. coli BL21(DE3) pLysS carrying either blaOXA-42 or blaOXA-43

The 900 bp blaOXA-42 and blaOXA-43 products from B. pseudomallei 1902a and E15 parent strains were ligated directly into pCR T7/CT-TOPO and transformed into E. coli TOP10F' as described in Materials and methods. One clone for each strain was selected for further study. The recombinant plasmids were extracted and transformed into E. coli BL21(DE3) pLysS.

ß-Lactam susceptibilities and ß-lactamase (prepared by periplasmic extraction) activities of the E. coli BL21(DE3) pLysS carrying the B. pseudomallei class D ß-lactamase genes were determined. The results are shown in Table 4. Extracts of E. coli BL21(DE3) pLysS carrying blaOXA-42 or blaOXA-43 displayed oxacillinase activity, which was not found in extracts of E. coli without plasmid or E. coli (pUC18). In contrast, E. coli BL21(DE3) pLysS carrying blaOXA-42 or blaOXA-43 did not show any resistance to ceftazidime or imipenem, and ceftazidime or imipenem hydrolytic activity was not detected in cell extracts.


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Table 4.  ß-Lactam susceptibilities and ß-lactamase activities of E. coli BL21(DE3) pLysS carrying either the blaOXA-42 or blaOXA-43 gene from B. pseudomallei
 
Expression of class D ß-lactamase in B. pseudomallei E15, parent and ceftazidime-resistant mutant strains

To examine whether the class D ß-lactamase is overexpressed in ceftazidime-resistant mutants, RT–PCR was performed on one parent strain and its resistant mutant progeny. B. pseudomallei strain E15 was chosen for this study because it was isolated from soil and hence is unlikely to have been exposed to ß-lactams at growth inhibitory concentrations. Total RNAs were isolated and quantified. Primers used in cDNA synthesis and RT–PCR are described in Materials and methods. The RNA from both the parent and the ceftazidime-resistant mutant strains was used to synthesize cDNAs, which were further used as templates in amplification. The predicted PCR product is 435 bp. The results showed clearly that the expression of the class D ß-lactamase gene is increased significantly in the ceftazidime-resistant mutant compared with the parent strain (Figure 4). This experiment was repeated three times and the results were consistent.



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Figure 4. Expression of class D ß-lactamases of B. pseudomallei E15, parent and mutant strains, by RT–PCR. Lane 1, 1 kb plus DNA ladder; lane 2, amplified product from E15 parent strain; lane 3, amplified product from E15 ceftazidime-resistant mutant (see Materials and methods for more details).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
ß-Lactam resistance poses a potential problem in the treatment of B. pseudomallei infection. ß-Lactam resistance can emerge in several ways, but the most important mechanism is the production of chromosomally mediated ß-lactamase(s). However, mutants in which ß-lactamase(s) are produced constitutively at high levels can occur naturally. In this study, ceftazidime-resistant mutants of B. pseudomallei were selected at high frequencies. This indicates that if B. pseudomallei is exposed repeatedly to ceftazidime, there exists the possibility that resistance will emerge rapidly.

Many ceftazidimases are plasmid encoded, arising as a result of mutations in TEM-, SHV- or the OXA-type ß-lactamases, leading to the extended-spectrum ß-lactamases (ESBLs). Livermore et al.9 reported ß-lactamases from B. pseudomallei that were strongly active against many cephalosporins, but not ceftazidime. However, specific ceftazidimase activity in B. pseudomallei was reported by Godfrey et al.10 The 25 B. pseudomallei tested in this study displayed ß-lactamase activities against different ß-lactams. The ability to hydrolyse ampicillin or cefalothin was not surprising, but the finding that 48% of B. pseudomallei strains tested possess the ability to produce ceftazidimase activity is worrying, as ceftazidime is the antibiotic of choice for the treatment of melioidosis.

The ability of bacteria to hydrolyse imipenem is often mediated by a metallo-ß-lactamase, although some serine enzymes have been reported to be able to hydrolyse carbapenems.6,23 Increased imipenemase activity (more than two-fold) in the B. pseudomallei mutant strains was found in 40% of those tested in this study. There is no direct evidence of a metallo-ß-lactamase gene in the B. pseudomallei genome currently being sequenced, but genome sequencing has revealed the presence of class A, C and D ß-lactamase genes. In P. aeruginosa, variants of class D ß-lactamases are recognized increasingly as being significant in resistance to ceftazidime, i.e. OXA-15,24 OXA-1821 and OXA-28.25 Some OXA-type enzymes have also been reported to hydrolyse carbapenems, e.g. OXA-23,26 OXA-24,7 OXA-25, OXA-26 and OXA-27.8 It was therefore decided to clone and sequence the class D ß-lactamase genes from B. pseudomallei strains 1902a and E15 and measure their substrate profiles to estimate the role of these enzymes in resistance to imipenem and ceftazidime.

The amino acid sequences of the two B. pseudomallei class D ß-lactamases, OXA-42 and OXA-43, are almost identical and show significant homology to chromosomally encoded oxacillinases from R. pickettii and Aeromonas spp. The presence of class D ß-lactamase seems to be ubiquitous among B. pseudomallei strains, as the geographical origins of these strains were diverse, and the organisms were isolated at different times (one was isolated 55 years ago). Of considerable interest, strain E15 possesses the class D ß-lactamase gene but was isolated from a rice field and has never been exposed to ß-lactams at therapeutic concentrations. These results support the possibility that the function of ß-lactamases is not only to inactivate ß-lactams but may also play some other role in cell physiology. If the ß-lactamases are involved in crucial cellular functions, then the genes will be maintained in these organisms.

The expression of blaOXA-43 is increased in the ceftazidime-resistant B. pseudomallei E15 mutant compared with its parent strain, as shown by RT–PCR. However, the results of ß-lactamase assays of extracts of E. coli carrying either blaOXA-42 or blaOXA-43 showed no detectable activities against ceftazidime or imipenem, so it is unlikely that overexpression of the class D enzyme is the reason for ceftazidime hydrolytic activity seen in some ceftazidime-resistant B. pseudomallei mutants. It should be noted, however, that there have been reports of oxacillinases associated with increases in MIC of ceftazidime up to 128 mg/L, even though they possess no detectable ceftazidimase activity, e.g OXA-16 from P. aeruginosa strains 906 and 961,27 and OXA-17 from P. aeruginosa strains 871 and 873.28 More commonly, however, resistance to third-generation cephalosporins is caused by the production of class A ESBLs29,30 or the overexpression of class C ß-lactamases.31 Class A and C ß-lactamases have already been shown to be involved in ß-lactam resistance in B. pseudomallei,32,33 so ceftazidime resistance may be caused by multiple ß-lactamases. Their relative roles now need to be elucidated.

The production of multiple ß-lactamases has been reported from many bacterial species. For example, expression of three ß-lactamases in Aeromonas spp. is co-ordinated despite the genes being unlinked.15,16,19 In the case of B. pseudomallei, the observed activities against different ß-lactam substrates as well as the results from genome sequencing suggest the presence of multiple ß-lactamases. Whether the expression of these enzymes is co-ordinated or not has yet to be determined, but the finding that the class D ß-lactamase is overexpressed in mutants selected for resistance to a ß-lactam most likely to be hydrolysed by one of the host’s other two ß-lactamases does suggest some co-ordination of ß-lactamase expression.


    Acknowledgements
 
We would like to thank Professor N. J. White, Wellcome Trust Unit, Bangkok, for supplying B. pseudomallei strains isolated in Thailand and the ceftazidime, and for critical reading of the manuscript. B. pseudomallei strains isolated from Vietnam and Australia were obtained from Dr Giles Hartman, Department of Pathology and Microbiology, University of Bristol. This work was funded by the Thailand Research Fund (TRF grant no. PDF/66/2543) and BSAC (grant no. 339). V.W. is supported by the Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme funded by the Wellcome Trust of Great Britain.


    Footnotes
 
* Corresponding author. Tel: +66-55-261000-4 ext. 4565; Fax: +66-55-261198; E-mail: pannikan{at}nu.ac.th Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . White, N. J. (2001). Clinical features of melioidosis. In Delegate Handbook and Abstract, World Melioidosis Congress, Perth, Australia, 2001. Abstract 6, p. 16. Australian Society for Microbiology, Melbourne, Australia.

2 . White, N. J., Dance, D. A., Chaowagul, W., Wattanagoon, Y., Wuthiekanun, V. & Pitakwatchara, N. (1989). Halving of mortality of severe melioidosis by ceftazidime. Lancet ii, 697–701.

3 . Dance, D. A., Wuthiekanun, V., Chaowagul, W. & White, N. J. (1989). The antimicrobial susceptibilities of Pseudomonas pseudomallei: emergence of resistance in vitro and during treatment. Journal of Antimicrobial Chemotherapy 24, 295–309.[Abstract]

4 . Dance, D. A., Wuthiekanun, V., Chaowagul, W., Suputtamongkol, Y. & White, N. J. (1991). Development of resistance to ceftazidime and co-amoxiclav in Pseudomonas pseudomallei. Journal of Antimicrobial Chemotherapy 28, 321–4.[ISI][Medline]

5 . Simpson, A. J., Suputtamongkol, Y., Smith, M. D., Angus, B. J., Rajanuwong, A., Wuthiekanun, V. et al. (1999). Comparison of imipenem and ceftazidime as therapy for severe melioidosis. Clinical Infectious Diseases 2, 381–7.

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