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
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Abstract |
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Introduction |
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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.
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Materials and methods |
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The clinical B. pseudomallei strains (1901a1928a) used in this study were isolated from patients in Ubon Ratchatani, north-east Thailand. Environmental strains (E10E188) were isolated from rice fields in the north-east of Thailand (Table 1).
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Ceftazidime-resistant mutants were selected by plating an overnight culture of B. pseudomallei onto ceftazidime-containing MuellerHinton 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 MuellerHinton 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 manufacturers 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 transcriptasePCR (RTPCR)
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 suppliers 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 RTPCR 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.
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Results |
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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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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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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 2324. Within the enzyme, the conserved Ser-Thr-Phe-Lys active site was found at position 5356. Four other conserved regions for class D ß-lactamases were also identified (Figure 1): Tyr-Gly-Asn (position 130132); Trp-Xaa-Gly-Xaa-Xaa-Leu-Xaa-Ile-Ser (position 149157), Gln-Xaa-Xaa-Xaa-Leu (position 161165); and Lys-Thr-Gly (position 201203).
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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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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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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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To examine whether the class D ß-lactamase is overexpressed in ceftazidime-resistant mutants, RTPCR 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 RTPCR 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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Discussion |
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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 RTPCR. 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 hosts other two ß-lactamases does suggest some co-ordination of ß-lactamase expression.
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Acknowledgements |
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Footnotes |
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References |
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