Department of Microbiology, Hospital Beaujon, 100 boulevard du Général Leclerc, 92110 Clichy, France
Received 3 September 2001; returned 17 January 2002; revised 6 February 2002; accepted 27 March 2002
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Abstract |
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Methods: Two hundred and fifty-nine P. aeruginosa isolates were screened by PCR with 11 primer pairs designed to detect genes encoding PSE, OXA, TEM and SHV enzymes. PSE and OXA gene variants were distinguished by restriction of PCR products with endonucleases recognizing sites involved in point mutations. Nucleotide sequences were verified for a few isolates by sequencing the PCR products.
Results: Four isolates produced extended-spectrum ß-lactamases (ESBLs) according to the double disc synergy test. PCR detecting blaPSE genes was positive in 162 (62.5%) isolates: 151 carried blaPSE-1 and 11 carried a variant encoding an enzyme differing from PSE-1 by a single amino acid substitution (Pro102 to Ser). PCR detecting sequences for enzymes of the OXA-10 group was positive in 68 (26.3%) isolates: 31 carried blaOXA-10, one carried blaOXA-14 and 36 carried a new variant intermediate between blaOXA-13 and blaOXA-19. The blaOXA-2 gene was identified in 13 (5%) isolates. Two other isolates carried blaOXA-2 variants encoding ESBLs differing from OXA-2 by a single amino acid substitution (Asp150 to Tyr and Trp159 to Cys, respectively). PCR detecting sequences for enzymes of the OXA-1 group was positive in 12 (4.6%) isolates. A blaTEM gene was identified in five (1.9%) isolates (three blaTEM-1, one blaTEM-2, one blaTEM-4).
Conclusion: This approach is effective for screening P. aeruginosa for ß-lactamase gene carriage in epidemiological studies and for detecting new variants.
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Introduction |
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Four PSE enzymes have been reported in P. aeruginosa: PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4. PSE-1, PSE-4 and CARB-3 are closely related, differing from one another by one or two amino acids, but share only 86.3% homology with CARB-4.36 Five distinct groups of oxacillinases have been described by Sanschagrin et al.7 OXA group I includes OXA-5, OXA-7, OXA-10 and its derivatives (OXA-11, OXA-14, OXA-16, OXA-17), and OXA-13 and its derivatives (OXA-19, OXA-28).817 OXA group II includes OXA-2, OXA-3, OXA-15 and OXA-20.7,1820 OXA group III includes OXA-1, OXA-4, OXA-30 and OXA-31, and OXA group IV is defined by OXA-9.7,2123 OXA group V currently consists of a single enzyme, LCR-1.7,9 In addition, OXA-18 does not belong to any of these groups and has very low amino acid identity with other oxacillinases.24
In previous studies that examined ß-lactam resistance mechanisms in a large number of P. aeruginosa isolates, characterization of ß-lactamases was based solely on isoelectric point.25,26 This method cannot identify these enzymes precisely, particularly those in the OXA group, since several enzymes have the same or very similar isoelectric points. We have therefore developed a method of identifying PSE and OXA ß-lactamase genes using PCRrestriction fragment length polymorphism (PCRRFLP) and applied it to characterization of the ß-lactamases produced in 259 clinical isolates of P. aeruginosa.
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Materials and methods |
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The different DNA sequences of genes encoding PSE and OXA enzymes from P. aeruginosa were analysed using DNA software analysis package Oligo 6 (Molecular Biology Insights, Cascade, CO, USA).324 Pairs of primers were designed to detect each group of ß-lactamases. Restriction endonuclease sites affected by point mutations were analysed and primers designed to distinguish variants within the same group.
Bacterial isolates
A collection of 259 clinical isolates of P. aeruginosa producing class A or D ß-lactamases was used for this study. The strains were isolated at Beaujon hospital during the periods 19841989 (128 isolates) and 19941999 (131 isolates), and were non-repetitive (only one isolate with a given antibiogram per patient was included in the set). Eighty-eight isolates belonged to serotype O12 and 171 to other serotypes. They were initially suspected of producing class A or class D ß-lactamases on the basis of routine MuellerHinton disc diffusion tests performed in our laboratory (resistance to ticarcillin, increased susceptibility to ticarcillin when combined with clavulanic acid and/or increased susceptibility to piperacillin when combined with tazobactam). Two hundred and twenty-five isolates were found to be susceptible to ceftazidime, while 34 isolates had decreased susceptibility to this drug. ß-Lactamase production was subsequently confirmed by detecting activity in sonicated extracts using the iodometric assay in an agar gel, with and without inhibitors (clavulanic acid, cloxacillin), according to Labia & Barthélémy.27 Isolates whose ß-lactamase activity was fully inhibited by cloxacillin, but not by clavulanic acid, were considered to produce only a derepressed class C cephalosporinase and were excluded from the study.
Detection of ESBLs
The 34 isolates with reduced susceptibility to ceftazidime were screened for the presence of ESBLs by a double disc synergy test. Three discs of ceftazidime were placed next to a disc of clavulanic acid at distances of 3, 2 and 1 cm. The test was considered positive when at least one of the three ceftazidime zones was expanded by the presence of clavulanic acid.
PCR screening
Bacterial DNA was prepared with InstaGene Matrix (Bio-Rad) according to the manufacturers instructions. DNA was used as template in PCR assays using 11 primer pairs: nine pairs, designed specially for this study, were based on the nucleotide sequences of blaPSE and blaOXA genes, and two pairs, reported previously by Rasheed et al.,28 detect blaTEM and blaSHV genes (Table 1). Reaction mixtures (50 µL) contained 50 mM KCl, 1.5 mM MgCl2, 0.5 µM of each primer, 0.4 mM of each deoxynucleotide triphosphate (Boehringer), 2.5 U of Taq polymerase (Roche Diagnostics) and 8 µL of DNA. The DNA amplification programme consisted of an initial denaturation step (96°C, 5 min) followed by 30 cycles of denaturation (96°C, 30 s), annealing (55°C or 60°C, 30 s) and extension (72°C, 1 min), and a single final extension of 5 min at 72°C. Ten microlitres of reaction mix containing PCR product was analysed by electrophoresis in 1.5% (w/v) agarose (Sigma).
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The blaPSE genes were identified by digestion of PCR products with ApoI and MboII (Figure 1). Genes encoding enzymes of OXA group I were differentiated by digestion of PCR products with BstEII, NdeI, Sau3AI, HaeIII, HhaI, BbvI and HphI (Figure 2). BbvI, HindIII and Sau96I were used to identify sequences encoding enzymes within the OXA group II (Figure 3). Ten microlitres of reaction mix containing PCR product was digested as instructed by the endonuclease supplier (New England Biolabs). The DNA fragments generated were analysed by electrophoresis in 24% (w/v) Metaphor agarose gel (FMC Products, Rockland, ME, USA), according to the expected fragment size.
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For at least one isolate corresponding to each type of PCRRFLP pattern observed, the identification of the ß-lactamase gene was verified by determining its sequence. Pairs of primers were used to amplify two overlapping fragments covering the full-length gene (Table 2) and the nucleotide sequences of the PCR products were determined by Genome Express on an Abiprism model 377 DNA sequencer (Perkin Elmer). The GenBank database was searched for homologous nucleotide sequences.
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Results |
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Among the 34 isolates with decreased susceptibility to ceftazidime, 29 were considered to overproduce the P. aeruginosa chromosomal class C cephalosporinase, in addition to producing a class A or D enzyme, since their ß-lactamase activities were inhibited only in the presence of both clavulanic acid and cloxacillin.25,27 Four isolates displayed a positive double disc synergy test; each of these was presumed to produce an ESBL. A synergy pattern was visible between the discs placed 3 cm apart for strain 606, 2 cm apart for strains 617 and 893, and only when placed 1 cm apart for strain 98. For the remaining isolate, which did not produce a derepressed cephalosporinase or an ESBL, ceftazidime resistance was presumed to be due to a non-enzymic mechanism.
PCR screening
Two hundred and fifty-eight of the 259 isolates tested gave positive PCR results with a single primer pair (161 with pair 1, 67 with pair 3, 13 with pair 4, 12 with pair 5, five with pair 10) and one isolate yielded PCR products with two primer pairs (pairs 1 and 3). In every case, the size of the PCR product was consistent with the predicted amplified fragment size (Table 1). None of the isolates gave positive PCR results for sequences encoding CARB-4, OXA-5, OXA-18, OXA-20, LCR-1 or the SHV enzyme group.
Identification of PSE group ß-lactamases
PCR with primer pair 1, which detects sequences for PSE-1, PSE-4 and CARB-3, was positive for 162 (62.5%) isolates. In all cases, ApoI cleaved the 321 bp amplification product, thus excluding CARB-3 (Figure 1). PSE-4 differs from PSE-1 by a C to A change at position 942, leading to the appearance of an MboII restriction site.4 Fragments generated by amplification with primer pair 13 were therefore subjected to MboII restriction. No cleavage of any of the 162 PCR products was observed, as expected for PSE-1. The nucleotide sequences of the ß-lactamase genes of three of these isolates were analysed and confirmed to be blaPSE-1 in two of them. The third gene differed from blaPSE-1 by a single C to T mutation at nucleotide 401 causing a Pro102Ser change in the amino acid sequence of the enzyme. This mutation generates a Tsp509I restriction site at position 398. The Tsp509I restriction patterns of the PCR products generated with primer pair 12 were analysed for the 162 isolates. The new Tsp509I restriction site was present in 11 of them. These 11 isolates were therefore assumed to carry a new blaPSE gene encoding a variant of PSE-1.
Identification of ß-lactamases of OXA group I
PCR with primer pair 3 detects genes encoding OXA group I enzymes.This reaction was positive in 68 (26.3%) isolates, including the ESBL-producing strain 98. BstEII did not cleave the 276 bp fragment obtained from any of the isolates, thereby excluding carriage of blaOXA-7. In 36 isolates, PCR products were cleaved to two fragments by NdeI but were not cleaved by Sau3AI, as expected for the blaOXA-13 PCR product (Figure 2). However, analysis of the nucleotide sequences of PCR fragments from three of these isolates showed that the bla genes differed from blaOXA-13 by a G to A mutation at position 1157, causing the replacement of Ser73 by asparagine in the ß-lactamase. This mutation involves the disappearance of a HhaI site. Accordingly, the HhaI restriction profiles of the PCR products generated with primer pair 18 were analysed. All 36 PCR products lacked the HhaI site, so it was concluded that these bla genes encode a variant of OXA-13.
For the remaining 32 isolates producing OXA group I enzymes (including strain 98), no digestion of the PCR products with NdeI was observed, as expected for blaOXA-10 and its derivatives. Digestion of PCR products with HaeIII and the absence of digestion with MboII identified the 31 non-ESBL genes as blaOXA-10. This was confirmed by determining the nucleotide sequence of the bla genes of two isolates. MboII digested the PCR product obtained with strain 98 whereas HaeIII did not. The PCR product also lacked the BbvI site typical of blaOXA-11 and the HphI site typical of blaOXA-16; so the bla gene was assumed to be blaOXA-14. This was confirmed by nucleotide sequence analysis.
Identification of OXA group II ß-lactamases
PCR analysis with primer pair 4, which detects genes encoding OXA group II ß-lactamases, gave products with 13 (5%) isolates, including the two ESBL-producing strains 617 and 893. In each case, the PCR product was cleaved by BbvI, but not by HindIII or Sau96I. The ß-lactamase genes in the 11 non-ESBL isolates were therefore presumed to be blaOXA-2 (Figure 3). This conclusion was confirmed by sequencing three of the PCR products.
The Sau96I recognition site typical of blaOXA-15 was not found in the PCR products from strains 617 and 893, suggesting the carriage of a blaOXA-2 variant. Analysis of the nucleotide sequences of these genes showed that the bla gene of strain 617 differs from blaOXA-2 by a G to T mutation at nucleotide 575 and from blaOXA-15 by a G to A mutation at nucleotide 576, resulting in a tyrosine at amino acid 150 instead of the aspartate found in OXA-2 and the glycine in OXA-15. The nucleotide sequence of the bla gene in strain 893 differs from blaOXA-2 by a G to T mutation at position 604, leading to the replacement of tryptophan by cysteine at amino acid 159.
Identification of OXA group III ß-lactamases
PCR analysis with primer pair 5, which detects genes encoding OXA group III enzymes, was positive for 12 (4.6%) isolates. Analysis of the nucleotide sequences of the PCR products from two of these isolates showed that in both cases the enzyme encoded was OXA-31.
Identification of TEM group ß-lactamases
PCR analysis with primer pair 10, which detects blaTEM genes, was positive for five (1.9%) isolates, including the ESBL-producing strain 606. Sequencing the PCR products revealed that three isolates harbour blaTEM-1, one harbours blaTEM-2 and strain 606 harbours blaTEM-4.
Distribution of ß-lactamases according to O serotype and study period
Assuming that the genes detected reflect the ß-lactamases produced, PSE-1 was identified in all isolates of serotype O12. Among isolates of other serotypes, 62 (36.3%) produced PSE-1, 108 (63.2%) produced another enzyme and one (0.5%) produced both PSE-1 and an OXA enzyme.
The comparative distribution of ß-lactamases during the two study periods is shown in Table 3. PSE-1 was predominant over both periods, followed by enzymes of OXA group I. OXA group II and TEM ß-lactamases and ESBLs were identified exclusively in isolates from 19941999, whereas most OXA group III ß-lactamases were found in isolates from 19841989.
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Discussion |
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As reported previously,26 so in this study PSE enzymes were the most common ß-lactamases, followed by oxacillinases. TEM enzymes were uncommon and none of the isolates produced a SHV enzyme. Within the PSE group, PSE-1 was by far the most common enzyme, and it was strongly associated with serotype O12. Thus, blaPSE-1 was found in all O12 isolates but in only 36.3% of other serotypes. Other carbenicillinases were not identified, but 11 isolates produced a variant of PSE-1 differing from it by a single amino acid (Ser102 instead of proline).
The OXA group I ß-lactamases described by Sanschagrin et al.7 included OXA-5, OXA-7 and OXA-10 and its variants. More recently, a new OXA-10-related subgroup, which includes OXA-13 and the ESBLs OXA-19 and OXA-28, has been identified.14,15,17 OXA-19 differs from OXA-13 by two amino acid substitutions (Ser73Asn and Gly157Asp). An aspartate at position 157 is also found in all OXA-10-derived ESBLs and appears to be important for ceftazidime hydrolysis.10,11,13,15 The amino acid at position 73 seems to influence the level of resistance to penicillins conferred. The presence of Ser73 in OXA-13 is unusual since asparagine is found at this position in most enzymes of the group.15 In the present study, analysis of the nucleotide sequences of the bla genes of three isolates revealed sequences encoding asparagine at position 73 and glycine at position 157, defining a new variant, intermediate between OXA-13 and OXA-19.
The OXA group III ß-lactamases include OXA-1 and its derivatives OXA-4, OXA-30 and OXA-31, which differ from OXA-1 by two, one and four amino acid substitutions, respectively.2123 Because the mutations responsible for the allelic variation do not create changes in restriction pattern that can be easily differentiated with commercially available endonucleases, the bla genes within this group could not be distinguished by PCRRFLP. Determination of the nucleotide sequences of the bla genes of two of the 12 isolates revealed blaOXA-31, encoding an enzyme described recently in a single isolate of P. aeruginosa.23
ESBLs were found in four isolates from 19941999. The double disc synergy test was positive when discs were placed 3 cm apart for the TEM-producing isolate, but the discs had to be brought closer to 2 cm or even 1 cm, to detect production of OXA-derived ESBLs. Thus, three of the four ESBLs would have been undetected by the standard disc test with a separation of 3 cm. One ESBL was correctly identified by PCRRFLP as OXA-14, an enzyme described previously in a P. aeruginosa isolate from Turkey. OXA-14 differs from OXA-10 by the replacement of Gly157 by aspartate.11 Two other ESBLs were OXA-2-derived enzymes. Prior to this report, OXA-15 was the only known extended-spectrum variant of OXA-2,19 differing from it by an Asp150Gly substitution. The mutation generates a Sau96I restriction site in the gene. Neither gene for the OXA-2-derived ESBLs had this site and were therefore presumed to have unknown point mutations, which were subsequently identified by nucleotide sequencing. The fourth ESBL was TEM-4, which has already been reported in a single isolate of P. aeruginosa.31
The limitation of this PCRRFLP approach is that it can only detect mutations at known positions. Analysing the nucleotide sequences of PCR products obtained from a few isolates fortuitously discovered point mutations at previously unknown positions, identifying new alleles encoding new enzyme variants. Some mutations may not involve the appearance or disappearance of a recognition site for commercially available endonucleases. These would not be detected by the current approach. Lastly, the method is rather time consuming and labour intensive because of the large number of PCRs and restriction digests needed to identify all ß-lactamases unambiguously. However, since a few enzymes are widespread in P. aeruginosa while others are very uncommon, in the majority of isolates, ß-lactamases can be detected and identified using four primer pairs (for the PSE and the three main OXA groups) and a limited number of endonucleases. This method can be augmented by sequence analysis of PCR products for ESBLs or enzymes conferring unusual resistance patterns.
In conclusion, PCRRFLP is an effective method of screening P. aeruginosa ß-lactamases in epidemiological studies. It gave results consistent with previous reports, which used isoelectric point analysis and indicated that PSE-1 is the most common acquired ß-lactamase in P. aeruginosa isolates, and revealed the distribution of various OXA enzymes in our hospital. Moreover, it was instrumental in the detection of four new ß-lactamase variants, including two OXA-2-derived ESBLs. Further studies are needed to clone and sequence the genes and describe the characteristics of these new ß-lactamases.
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Footnotes |
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References |
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