JAC Advance Access originally published online on July 1, 2006
Journal of Antimicrobial Chemotherapy 2006 58(3):530-536; doi:10.1093/jac/dkl266
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Characterization of acquired ß-lactamases and their genetic support in multidrug-resistant Pseudomonas aeruginosa isolates in Taiwan: the prevalence of unusual integrons
1 Departments of Pathology, College of Medicine, National Cheng Kung University No. 1 University Road, Tainan 70101, Taiwan 2 Internal Medicine, College of Medicine, National Cheng Kung University No. 1 University Road, Tainan 70101, Taiwan 3 Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University No. 1 University Road, Tainan 70101, Taiwan 4 Department of Laboratory Medicine, National Taiwan University Hospital No.7 Chung San South Road, Taipei 10002, Taiwan 5 Departments of Pathology, Tri-service General Hospital Sec. 2, 325 Chenggong Road, Taipei 11490, Taiwan 6 Internal Medicine, Tri-service General Hospital Sec. 2, 325 Chenggong Road, Taipei 11490, Taiwan
*Corresponding author. Tel: +886-6-2353535 ext. 5605; Fax: +886-6-2363956; E-mail: jjwu{at}mail.ncku.edu.tw
Received 10 March 2006; returned 2 May 2006; revised 19 May 2006; accepted 1 June 2006
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Objectives: The present study was conducted to investigate acquired ß-lactamases and their genetic support in 26 Pseudomonas aeruginosa isolates that were resistant to nearly all antipseudomonal drugs from six medical centres in Taiwan.
Methods: Acquired ß-lactamases and their genetic support were determined by PCR-based strategies.
Results: Four and 16 of the 26 isolates were found to produce VIM-2 and VIM-3 metallo-ß-lactamases (MBLs), respectively, and 1, 1 and 2 isolates produced OXA-17, OXA-10 and PSE-1, respectively. These bla genes are all in class 1 integrons that are probably chromosomally located. The blaVIM-3-containing integron, with a deletion between int1 and the blaVIM-3 structural gene, has six gene cassettes, blaVIM-3, a probable fosfomycin resistance determinant, aacA4, aacA4, aadB and aacA4. The blaVIM-2-containing integron, without detectable 5'-conserved segment, contains four genes cassettes (aacA7-blaVIM-2-dhfr-aacA5) and is ended by tniC. The blaOXA-10-containing integron includes a catB3 cassette and a fused gene cassette, which is made up of blaOXA-17 and a novel streptomycin–spectinomycin gene, designated aadA15. The blaOXA-17-containing integron has three gene cassettes (aacA4-catB2-blaOXA-17) but the 59-base element of the blaOXA-17 cassette is interrupted by a putative transposase gene. The blaPSE-1-containing integron has three gene cassettes, aacA4, an aadA3-related gene designated aadA3b and blaPSE-1. PFGE revealed genetic diversity among the multidrug-resistant isolates from different hospitals.
Conclusions: This study demonstrated the high prevalence of VIM-type MBLs and the presence of unusual bla-encoding integrons in multidrug-resistant P. aeruginosa isolates in Taiwan. The spread of blaVIM-2-related genes by horizontal transfer might have occurred.
Keywords: carbapenemases , penicillinases , gene cassettes , fosfomycin
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Carbapenems play an important role in the treatment of infections caused by Pseudomonas aeruginosa, and the increasing frequency of carbapenem-resistant P. aeruginosa isolates is of concern worldwide.1,2 Carbapenems can be hydrolysed by Ambler's class B ß-lactamases and a few class A and D ß-lactamases.3–6 Four major groups of acquired class B ß-lactamases, also named metallo-ß-lactamases (MBLs), have been identified; they are IMP-, VIM-, SPM- and GIM-type ß-lactamases.4 Among carbapenemases, the IMP- and VIM-type MBLs occur most widely and commonly in Gram-negative bacilli including P. aeruginosa.4,7–13 The number of reported IMP and VIM variants is increasing,4 and there are at least 14 and 21 nucleotide sequences of VIM and IMP variants, respectively, deposited in the GenBank database. Almost all MBL genes were found as gene cassettes in integrons, class 1 integrons in particular.4,7–9,13–16
In Taiwan, several VIM- and IMP-type MBLs have been described,10,17,18 among which, only a single VIM variant, VIM-3, has been identified in P. aeruginosa.10 The amino acid sequence of VIM-3 differs from that of VIM-2, the most widely spread VIM-type MBL, by two amino acid substitutions.10 VIM-3 is probably chromosomally encoded, and the genetic environment of blaVIM-3 remains unknown. Non-MBL carbapenemases have not been detected in Taiwan. More recently, the emergence of P. aeruginosa isolates resistant to nearly all clinically available antipseudomonal drugs was noticed in a Taiwanese university hospital, and their spread may become a serous threat to hospitalized patients.19,20 The present study was conducted to investigate ß-lactamase contents in such multidrug-resistant P. aeruginosa (MDRPA) isolates from six medical centres in Taiwan. The genetic environments of acquired ß-lactamase genes were also investigated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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MDRPA isolates
A total of 26 MDRPA isolates, defined as isolates intermediately resistant or resistant to antipseudomonal penicillin/ß-lactamase inhibitor combinations and all antimicrobial agents available for clinical use against P. aeruginosa (cephalosporins, aztreonam, carbapenems, aminoglycosides and ciprofloxacin), were collected between March and August 2003 from six medical centres in Taiwan. Each isolate was from a unique patient and was phenotypically characterized on-site by the standard disc diffusion method.21
Susceptibility testing
MICs of piperacillin–tazobactam, ticarcillin–clavulanic acid, ceftazidime, cefepime, aztreonam, imipenem, meropenem, ciprofloxacin, gentamicin and amikacin were determined by the agar dilution method according to the CLSI (formerly NCCLS) guidelines.22 MICs of fosfomycin for isolates and transformants in the cloning experiments described below were determined by the Etest method (AB BIODISK, Solna, Sweden) according to the manufacturer's instructions.
Screening of MBL producers
MDRPA isolates were screened for MBL production using the 2-mercaptopropionic acid double-disc potentiation method as described previously.23 Enhancement of the zone of inhibition in the area between a 2-mercaptopropionic acid disc and any one of four discs containing ceftazidime, ceftazidime–clavulanate, cefepime and cefepime–clavulanate suggests MBL production.
ß-Lactamase assays
Crude preparations of ß-lactamases were obtained by sonication as described previously.24 We performed analytical isoelectric focusing (IEF) by the method of Matthew et al.25 with an LKB Multiphor apparatus (GE Healthcare Life Sciences, Hong Kong, China) on prepared PAGplate gels (pH 3.5–9.5; GE Healthcare Life Sciences). ß-Lactamase activity was detected by overlaying the gels with 0.5 mM nitrocefin (Oxoid, Basingstoke, UK) in 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (pH 7.5) supplemented with 2 mM ZnCl2.10,14 Hydrolysis of 0.1 mM imipenem (Merck Sharp & Dohme, Rahway, NJ, USA) by crude preparations of ß-lactamases was monitored by a Beckman DU-7 ultraviolet spectrophotometer (Palo Alto, CA, USA) at 297 nm and 37°C.10,14 Inhibition of enzyme activity was determined by measuring the residual carbapenemase activity after incubation of the crude extract with 10 mM EDTA or 50 µM clavulanic acid for 20 min at 25°C.
PCR amplification and DNA sequencing
Genomic DNA was extracted with a commercial kit (QIAGEN, Valencia, CA, USA). The presence of bla genes was detected by PCR with primers pse-1-1A and pse-1-1B for blaPSE-1 and with the previously reported primers for blaVIM-1-, blaVIM-2-, blaIMP-1-, blaIMP-2- and blaOXA-10-related genes (Table 1).10,26 Association of integrons with ß-lactamase gene was investigated by PCR using primers for bla detection and integron-specific primers (Table 1 and Figure 1). The amplicons were purified with a PCR clean-up kit (Roche Applied Science, Mannheim, Germany) and both strands of them were sequenced with the primers for PCR or by gene walking with custom sequencing primers. Nucleotide and amino acid sequences were analysed and compared by use of the BLAST computer program (National Center for Biotechnology Information) and the FASTA program (Genetics Computer Group).
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PFGE
PFGE of SpeI-digested genomic DNA samples of MDRPA isolates was carried out with a CHEF-DR 3 apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the instruction manual. PFGE patterns were interpreted in accordance with the criteria of Tenover et al.27
Hybridization assays
Plasmids from bacterial isolates were prepared by a rapid alkaline lysis procedure.28 EcoRI-digested genomic DNA samples and undigested plasmids were electrophoresed on 0.8% agarose gels and then subjected to Southern hybridization. The bla genes amplified by PCR were labelled with digoxigenin (Roche Applied Science). Labelling and detection of the probes were carried out using a DIG DNA labelling and detection kit (Roche Applied Science) according to the manufacturer's instructions.
Cloning experiments
The entire blaVIM-3 and its following gene were amplified by a PCR technique using primers int1-8A, which hybridized to the upstream region of the two potential promoters for the class 1 integron and aacA4-2B (Table 1). The amplicon was inserted into pGEM-5Zf (+) (Promega, Madison, WI, USA) and cloned into Escherichia coli JM109 (Promega). The sequence of the insert was verified by nucleotide sequencing.
Nucleotide sequence accession numbers
Nucleotide sequences for integrons from isolates N2-15, N2-11, C-34 and N2-1 were submitted to GenBank under accession no.s DQ393781 [GenBank] –DQ393784 [GenBank] .
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Detection of ß-lactamases
Among the 26 MDRPA isolates, 20 (76.9%) isolates were suggested to produce MBLs by the 2-mercaptopropionic acid double-disc method. The 6 MBL non-producers showed no discernible carbapenemase activity in the hydrolysis assay (data not shown). In the IEF analysis, each isolate produced a pI > 8.5 ß-lactamase, which was consistent with the intrinsic AmpC enzyme.3 Moreover, 16 and 4 of the 20 putative MBL producers expressed a pI 5.3 ß-lactamase and a pI 5.4 ß-lactamase, respectively. PCR and nucleotide sequencing analyses indicated that the pI 5.3 and 5.4 ß-lactamases were consistent with VIM-2 and VIM-3 MBLs, respectively. The presence of blaVIM-2 in P. aeruginosa was detected for the first time in Taiwan. Among the 6 non-MBL producers, two isolates expressed a pI 5.7 ß-lactamase and two isolates expressed a pI 6.1 ß-lactamase. PCR and nucleotide sequencing revealed the presence of blaPSE-1 in both isolates with a pI 5.7 ß-lactamase and the presence of blaOXA-10 or blaOXA-17 in the two isolates with a pI 6.1 ß-lactamase.3,29 Two of the 26 MDRPA isolates showed the expression of putative intrinsic AmpC only on the IEF gel. The presence of blaIMP-1-, blaIMP-2- and blaVIM-1-related genes was not detected by PCR in all 26 MDRPA isolates.
Susceptibility testing
All 26 MDRPA isolates were resistant to piperacillin–tazobactam (MIC 
128 mg/L) and ticarcillin–clavulanic acid (MIC 128 mg/L). MICs of other antimicrobials are summarized in Table 2.
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PFGE analysis
PFGE of SpeI-digested genomic DNA samples revealed 16 major patterns among the 26 MDRPA isolates (Table 2). The 4 blaVIM-2-positive and 16 blaVIM-3-positive isolates gave 3 and 7 major patterns, respectively. No interhospital clonal dissemination of MDRPA isolates was found but intrahospital dissemination of closely genetically related VIM-2- or VIM-3-producing isolates was observed.
Structures of bla-containing integrons
The presence of the int1 gene was detected by PCR with primers Int1-1A and Int1-1B in 23 MDRPA isolates. One blaPSE-1-positive isolate (isolate S2-40) and the two isolates without detectable acquired ß-lactamases gave a negative result. Sequence analysis of PCR products from selected isolates (isolates N2-1, S1-36, C-34, N2-11 and N2-15) confirmed the sequences of int1.
The primers Int1-6A and Vim2-1B generated an 
1 kb fragment and the primers Vim2-3A and qacE-2B generated an 3.3 kb fragment from all 16 blaVIM-3-positive isolates. Both amplicons from each isolate were digested with HaeIII. All isolates showed the same restriction patterns, suggesting that all VIM-3-producing isolates were identical in possessing a blaVIM-3-containing integron. Sequence analysis of the amplicons from isolate N2-1 revealed that blaVIM-3 is in an unusual integron containing 6 gene cassettes (Figure 1a). Comparison of the 5'-conserved segment (5'-CS) and the blaVIM gene cassette between the blaVIM-3 integron and In56 (AF191564
[GenBank]
), a blaVIM-2-containing integron identified from a P. aeruginosa isolate from France,16 revealed a deletion of 65 bp sequence upstream of the blaVIM-3 start codon in the blaVIM-3 integron. The deleted region includes a part of attI1 and the core site of the blaVIM-3 cassette. Since the full attI1 site is required for high-efficiency recombination with a 59-base element (59-be),30 it is very likely that the deletion occurred after the intact blaVIM-3 gene cassette had been inserted into the integron and it has now become fused in place. Downstream of blaVIM-3 is a 402 bp open reading frame (ORF), designated orf2. The deduced amino acid sequence of orf2 is most closely related to the orf ‘I’ product (74.3%) from a Korean P. aeruginosa strain YMC95/1/704 (AY029772
[GenBank]
),8 and is similar to those of known fosfomycin resistance determinants, including FosA (34.5% identical) in the composite transposon Tn2921 in Serratia marcescens (M85195
[GenBank]
),31 FosB (34.7% identical) in the plasmid from Staphylococcus epidermidis (X54227
[GenBank]
),32 PA1129 (35.7%) in the chromosome of P. aeruginosa PAO1 (AE004543)33 and ORF1 (39.5%) in P. aeruginosa Mß-7 (AY294333
[GenBank]
).7 Thus, it is likely that orf1 is also a fosfomycin resistance determinant.
The primers Int1-6A and Vim2-1B generated an 1.7 kb fragment from all 4 blaVIM-2-positive isolates. No amplification was observed by PCR with primers Vim2-3A and qacE-2B. Instead, an 1.5 kb fragment was amplified with the primers Vim2-3A and tniC-1B from each of the four isolates. The primer tniC-1B was located within the tniC structural gene, which was considered to code for a recombination protein. The PCR products generated by the two primer pairs were digested with HaeIII. All four blaVIM-2-positive isolates showed the same restriction patterns, suggesting that the blaVIM-2 genes in the four isolates were surrounded by the similar genetic environments. Sequence analysis of the amplicons from isolate S1-36 revealed that blaVIM-2 is located in an unusual integron (Figure 1b), of which the sequence is 100% identical to that of a blaVIM-2-containing integron from a P. aeruginosa isolate from the United States (AY943084
[GenBank]
).9 The presence of such an unusual integron in isolates from different countries is interesting but the epidemiological link is unclear.
The primers Int1-6A and OPR-2 generated an 1.7 kb fragment and the primers OPR1 and qacE-2B generated an 1.8 kb fragment from the blaOXA-10-positive isolate. Sequence analysis revealed that blaOXA-10 is located in an integron containing a catB3 cassette and a fused gene cassette (Figure 1c). The fused gene cassette is made up of the blaOXA-10 cassette and a cassette with a novel streptomycin–spectinomycin resistance gene, designated aadA15. The coding protein (263 amino acids) of aadA15 is most closely related to those of aadA12 (AY665771
[GenBank]
; 96% identical) and aadA1 (M95287
[GenBank]
; 95% identical).34 blaOXA-10 is separated from aadA15 by a 16 bp region, of which the sequence is 100% identical to the sequence between blaOXA-10 and aadA1 in the fused gene cassette, blaOXA-10/aadA1, in In53 (AF205943
[GenBank]
), an unusual integron encoding the VEB-1-type extended-spectrum ß-lactamase (ESBL) from an E. coli isolate from France,35 and the sequence of 59-be of the fused cassette is 100% identical to that of the blaOXA-10/aadA1 cassette. Thus, the fused blaOXA-10/aadA15 cassette may have developed by the fusion of two gene cassettes via a process of 59-be deletion similar to that in In53 or by point mutations in the aadA1 structural gene after the fusion of blaOXA-10 and aadA1 cassettes.
The primers Int1-6A and OPR-2 generated an 2.4 kb fragment and the primers OPR1 and qacE-2B generated an 2.3 kb fragment from the blaOXA-17-positive isolate. Sequence analysis revealed that blaOXA-17 is located in an integron containing three gene cassettes, aacA4, catB2 and blaOXA-17 (Figure 1d). The 59-be of the blaOXA-17 cassette is interrupted by a 1059 bp ORF, designated orf4. The deduced amino acid sequence of orf4 is 40% identical to that of TnpA encoded by In109 (AY920928
[GenBank]
), an integron encoding the GES-type ESBL from a French P. aeruginosa isolate,36 suggesting that orf4 also encodes a transposase. The blaOXA-17/orf4 cassette terminated at a distance of only 7 bp from the start codon of qacE
1. Sequence analysis suggests the occurrence of a deletion between the 59-be of the blaOXA-17 cassette and the qacE1 structural gene [5' end of the 3'-conserved segment (3'-CS)].
The primers Int1-6A and Pse-1-1B generated an 2.7 kb fragment and the primers Pse-1-1A and qacE-2B generated an 1.1 kb fragment in both blaPSE-1-positive isolates. The sequence analysis of the PCR products revealed that blaPSE-1 in each blaPSE-1-positive isolate is located in an integron containing three gene cassettes (Figure 1e). Upstream of the blaPSE-1 cassette is a gene cassette containing a novel streptomycin–spectinomycin resistance gene, named aadA3b. aadA3b has only two nucleotide differences from aadA3 (AF047479
[GenBank]
),37 leading to His to Gln and Ile to Val changes at amino acid positions 245 and 250, respectively. Since no amplification was observed by PCR with the primers Int1-1A and Int1-1B, point mutations or a deletion may have occurred in the int1 gene of the integron from isolate S2-40.
Hybridization assays
After plasmid extraction, no plasmids were detected by agarose gel electrophoresis for all 26 MDRPA isolates, and the blaVIM-3, blaOXA-10 and blaPSE-1 probes hybridized with the bands of possible chromosomal DNAs from the blaVIM-, blaOXA- and blaPSE-1-positive isolates, respectively. Thus, it is likely that each of these bla genes was on the chromosomes. In the hybridization analysis of the EcoRI-digested chromosomal DNA samples, the blaVIM-3 probe hybridized with a 4.5 kb fragment in all blaVIM-2-positive isolates and with a 8.8 or 6.2 kb fragment in all blaVIM-3-positive isolates except one (Table 2 and Figure 2). The result suggests the occurrences of horizontal transfer of the blaVIM-containing integrons among different strains. The present study did not confirm the chromosomal locations of the bla genes. If this can be proved, how the bla-containing integrons were mobilized between chromosomes would become an interesting and important issue. Further studies will be performed to elucidate these problems.
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Cloning experiments
Including the entire blaVIM-3 and orf2 genes, the 1753 bp PCR product obtained with primers int1-8A and aacA4-2B was cloned into pGEM-5Zf (+), and E. coli JM109 was transformed with the recombined plasmid. The sequence of the inserted fragment was verified by nucleotide sequencing. The transformant was tested by the double-disc screening method for MBL production and gave a positive result. The transformant was resistant to ceftazidime (MIC 64 mg/L) and susceptible to imipenem (MIC 0.25 mg/L) in the agar dilution test. The result is consistent with the previous observation that the cloned IMP or VIM MBLs do not confer resistance to carbapenems in E. coli.14–17 Moreover, 6 MDRPA isolates did not possess ß-lactamases associated with carbapenem resistance. Thus, other mechanisms such as hyperexpression of efflux pumps may play an important role in the carbapenem resistance of our MDRPA isolates.38,39 Although orf2 is suggested to be a fosfomycin resistance determinant by sequence analysis and all VIM-3-producing P. aeruginosa isolates appeared resistant to fosfomycin (MIC > 256 mg/L), the E. coli transformant was susceptible to this drug (MIC 1 mg/L). Two explanations are possible: either the orf2 gene is not fully expressed or the coding protein has minimal activity against fosfomycin. The function of the protein needs further biochemical characterization.
This report describes the high prevalence of VIM-type MBL among MDRPA isolates, the presence of unusual integrons encoding ß-lactamases, and the detection of two novel gene cassettes containing streptomycin–spectinomycin resistance genes in Taiwan. Two VIM subtypes were identified, and VIM-2 was detected in P. aeruginosa for the first time in Taiwan. Genetic diversity of MBL producers, the presence of similar integrons demonstrated by restriction mapping and the same restricted fragments hybridized with the blaVIM probe suggest the occurrences of horizontal transfer of the blaVIM-2 and blaVIM-3-containing integrons in P. aeruginosa. Since the spread of MDRPA isolates may become a serious clinical problem in Taiwan, further surveillance and characterization of MDRPA isolates should be a high priority.
Transparency declarations
None to declare.
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Acknowledgements |
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We thank Jainn-Ming Shyr at Taichung Veterans General Hospital, Taichung, Taiwan, Yin-Ching Chuang and Yi-Chueh Yang at Chi-Mei Medical Center, Tainan, Taiwan, Li-Shin Wang at Buddhist Tzu-Chi General Hospital, Hualien, Taiwan and Chien-Fang Peng at Kaohsiung Medical University, Kaohsiung, Taiwan for providing MDRPA isolates.
This work was funded by a grant NSC 94-2320-B-006-048 from the National Science Council, Taiwan.
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