a Department of Clinical Pathology, Showa University Fujigaoka Hospital, 1-30 Fujigaoka, Aoba-ku, Yokohama 227-8501; b Department of Biochemistry Sagami Women's University, 2-1-1 Bunkyou, Sagamihara 228-0807; c Department of Biochemistry, Division of Pharmaceutical Science, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
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Abstract |
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Introduction |
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Pseudomonas aeruginosa causes nosocomial infections and is resistant to many antimicrobial agents. By examin-ing 186 isolates of P. aeruginosa collected at Showa University Fujigaoka Hospital between 1992 and 1993, we found 28 isolates of P. aeruginosa that did not show the normal correlation between resistance to cefclidin (E1040, an alkoxyiminocephalosporin with aminothiadiazol and carbamoylquinuclidino residues) and resistance to ceftazidime. These isolates were resistant to cefclidin (with MICs in the range 25100 mg/L) and susceptible to ceftazidime (with MICs in the range 3.1312.5 mg/L).4
In this study, the pIs of the ß-lactamases from 22 of the 28 isolates were determined, after the purified ß-lactamase from strain FHPA1105 (one of the isolates) had been characterized. The genes were analysed by using single-strand conformation polymorphism (SSCP) and Southern hybridization based on the polymerase chain reaction (PCR) method.
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Materials and methods |
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A total of 186 isolates of P. aeruginosa were collected at Showa University Fujigaoka Hospital between 1992 and 1993. 4 Of these isolates, 28 were resistant to cefclidin but not to ceftazidime; six of these died during storage at 80°C and the remaining 22 (listed in the legend of Figure 1) were used in this study. Escherichia coli ML4901 harbouring the plasmic pRms213 (encoding the OXA-1 enzyme) and E. coli C600 harbouring the plasmid pMG203 (encoding the OXA-4 ß-lactamase) were used as reference strains. These two ß-lactamase-producing strains were kindly provided by Dr Shizuko and Dr George A. Jacoby, respectively.
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Bacteria were grown to late log phase, harvested, sonicated and centrifuged at 100,000g at °C for 30 min. The supernatants were used as a crude extract. The crude extract of strain FHPA1105 or the E. coli ML4901 was precipitated by adding 40 80% (w/v) ammonium sulphate, and the solution was dialysed against 50 mM sodium phosphate buffer, pH 6.0. The enzyme was partially purified using CM-Sephadex C-50 and Sephadex G-75 column chromatography. The enzyme from strain FHPA1105 was further purified using Mono Q column chromatography (FPLC, Pharmacia, Uppsala, Sweden).
ß-lactamase assay
All assays were conducted using a Hitachi 228 spectrophotometer (Hitachi Industrial Co., Tokyo, Japan) with a heated attachment at 30°C. The wavelengths (nm) and absorbance differences (mM-1 cm-1) obtained by measuring the hydrolysis of cefclidin, ceftazidime and piperacillin were 265 and 21.6, 257 and 22.9, and 232 and 11.1, respectively. The initial rate of hydrolysis was determined at a substrate concentration of 100µM. One unit of activity was defined as the amount of enzyme that hydrolysed 1µmol of substrate per minute at 30°C in 50 mM sodium phosphate buffer, pH 7.0. Enzyme activity was standardized against the protein concentration measured by the method of Bradford. 5
Kinetic parameters of the partially purified enzyme from strain FHPA1105 for ß-lactam antibiotics were determined using the method of Lineweaver & Burk 6 and compared with the reference OXA-1 ß-lactamase.
The inhibitory effect of clavulanic acid upon the enzyme was expressed as the concentration (IC50) of the inhibitor that was required to inhibit the cephaloridine-hydrolysing activity by 50%. 7 The inhibitory effect of EDTA was expressed as the cephaloridine-hydrolysing activity of the enzyme in the presence of EDTA (1 mM) relative to the activity in its absence. 7
SDSpolyacrylamide gel electrophoresis (SDS PAGE)
The molecular mass of the ß-lactamases was estimated by SDS PAGE on a 12.5% gel by the method of Laemmli. 8
Isoelectric focusing
Isoelectric focusing of ß-lactamases was performed using a 5% polyacrylamide (acrylamide:bis, 9.7:0.3) slab gel containing 6% ampholine (pH range 3.510.0) (Pharmacia) at 4°C for 1.5 h (anode solution, 0.2 M H3; PO4; cathode solution, 1 M NaOH) at 15 W of constant power on a flat apparatus (Atto Co., Tokyo). The pIs were determined using a Separax sheet overlay (Jookou Co., Tokyo) that had been soaked in nitrocefin (0.5 g/L).9 To calibrate the assay, a calibration kit for determining the pI of proteins within the pH range 3.59.3 (Pharmacia) was used.
N-terminal sequencing
The 21 N-terminal amino acid residues of the purified enzyme from strain FHPA1105 were determined with an automated gas-phase sequencer (Model 470, Applied Biosystems). The EMBL and GenBank databases were searched for homologous amino acid sequence.
PCR method
A bacterial suspension, in deionized water, was boiled for 20 min, then filtered through Millipore filters (pore size, 0.22µm). Next, 0.1% SDS and 200µg proteinase K (Sigma, St Louis, MO, USA) were added to the solution and the mixture was incubated at 37°C for 1 h. Template DNA was extracted by the phenol/chloroform method. 10 The primer pairs (forward and reverse) and the positions of the amplified products for the PCR method (Table I) were designed based on the complete nucleotide sequence of OXA-1 precursor ß-lactamase (preOXA-1). 11 Each PCR contained 10µL of template DNA, 1 µM of each primer, 10µL of concentrated buffer, 8µL of 2.5 mM dNTP, 8 µL of 25 mM MgCl2; and 1µL of Taq DNA polymerase (5 units/µL) (Greiner Japan, Tokyo); deionized water was added to give a final volume of 100µL. The amplification reaction consisted of 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 51°C, and polymerization for 2 min at 72°C for the primer pairs ( Table I).
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The PCR products were denatured by adding an equal volume of denaturing solution (23.75 mL of 99% formamide, 1.25 mL of 1% xylene cyanol solution and 10 mg of bromophenol blue in a total volume of 25 mL of total volume) at 95°C for 5 min and thereafter directly placed in ice/ ethanol to prevent reannealing of the single-stranded products. Electrophoresis was performed with a CleanGel DNA Analysis Kit (Pharmacia) at 2.5 W for 10 min at 15°C and then at 9 W for about 45 min (until the bromophenol blue had reached the anode), and the DNA was strained with the PlusOne DNA Silver Staining Kit (Pharmacia).
Southern hybridization method
Each DNA lysate, containing both plasmid and chromosomal DNA, was concentrated by adding 10% (w/v) polyethylene glycol (PEG) 6000 and 0.5 M NaCl, and was extracted by the phenol/chloroform method. The DNA was blotted on to a nylon membrane for 18 h. The full-length gene of the reference preOXA-4 enzyme was amplified by PCR and labelled with digoxigenin. Hybridization was performed under high-stringency conditions. The DNA hybrids were detected using the DIG High Prime DNA Labelling and Detection kit I for chemiluminescent detection with CSPD (Boehringer Mannheim, Mannheim, Germany). The exposure time on Polaroid film 612 was 30 or 50 min.
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Results |
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The purified ß-lactamase from strain FHPA1105 was inhibited by clavulanic acid (IC50 = 0.5µM), but not by 1 mM EDTA. The molecular mass was 33 kDa according to SDS PAGE. The N-terminal amino acid sequence was STDISTVASPLFEGTEGCFLL. A search of the EMBL and GenBank databases revealed that this amino acid sequence was consistent with that of the OXA-1 type of ß-lactamase. 11 However, the N-terminal sequence of the OXA-4 ß-lactamase was not registered with either database. The kinetic parameters of the purified enzyme for benzylpenicillin, ampicillin, carbenicillin, cloxacillin, piperacillin, imipenem, cephaloridine, cephalotin, cefotaxime, cefrazidime, cefclidin and aztreonam were very similar to those of the reference OXA-1 ß-lactamase (data not shown). Since the various types of ß-lactamase are classified based on their pIs, 7 the pIs of the ß-lactamases from the 22 dissociated isolates in this study were determined ( Figure 1). All isolates produced ß-lactamases with pIs corresponding to that of the reference OXA-4 ß-lactamase (pI 7.5) rather than the reference OXA-1 ß-lactamase (pI 7.4). These crude extracts of the 22 isolates and the reference strains producing OXA-1 and -4 enzymes had cefclidin- and piperacillin-hydrolysing activities in the ranges 1436 and 2558 units/g, respectively. However, they had no ceftazidime-hydrolysing activities. These results confirm that these isolates produce an OXA-4 ß-lactamase.
PCR-SSCP
The amino acid sequence of the OXA-4 ß-lactamase has been reported
3 but the corresponding nucleotide sequence was not shown.
The complete sequence of the gene encoding the OXA-1 ß-lactamase has been determined,
11 so primer pairs were designed based on this nucleotide
sequence (Table I). The template DNAs of the 22 isolates were amplified
by primer pair 1, covering the full-length gene of the preOXA-1 ß-lactamase. All PCR
products amplified were 0.84 kb in length, which was consistent with the molecular size of the
reference preOXA-1 and -4 genes. SSCP was performed with the products amplified by various
primer pairs covering two point mutations, A928T and T1406
G or A, corresponding
to the amino acid substitutions Asp48
Val and Asp20
RGlu, respectively (
Figure 2). There was a marked difference in the SSCP patterns between
the PCR
products of the reference OXA-1 and -4 genes amplified by the primer pair 3 covering one point
mutation at A928
T. In contrast, the SSCP patterns of the products amplified by the other
primer pairs (2, 4 and 5) were equivalent in both reference genes, showing that these primers
could not be used to distinguish the two genes (Figure 2). The SSCP
patterns of the amplified products from the 22 isolates were therefore analysed using primer pair
3. The SSCP patterns of these ß-lactamase genes were consistent with that of the reference
OXA-4 gene, rather than reference OXA-1 gene (Figure 3).
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Since the OXA-4 ß-lactamase is plasmid-mediated,
1,12 the 22 isolates
were analysed by Southern hybridization based on the PCR method, to determine whether they
produced the OXA-4 ß-lactamase (Figure 4a). Total DNA
(600800 ng) from the 22 isolates was electrophoresed. The chromosomal DNA, but not
the plasmid DNA, from these isolates hybridized with the amplified preOXA-4 gene probe from
the reference E. coli C600. The plasmids pMG203 and pRms213 as well as the
chromosomes from the reference strains hybridized with the probe. The hybridization sensitivity
of detecting the plasmid encoding OXA-4 ß-lactamase was 103 ng of total DNA from
the reference E. coli C600 prepared by concentration with polyethylene glycol (Figure 4b). A 47 kb plasmid and the chromosomal DNA were separated
from the total DNA of strain FHPA1105, but this plasmid did not hybridize with the OXA-4
gene probe (Figure 5). These findings show that in these 22 isolates the
OXA-4 gene is borne on the chromosome and not on a plasmid.
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We evaluated the distribution of P. aeruginosa producing OXA-4 ß-lactamase in the hospital. The distribution of the P. aeruginosa isolates according to hospital wards and clinical specimens is shown in Table II. The 28 isolates that were resistant to cefclidin but not to ceftazidime (including the six isolates that died in storage, which probably produced the OXA-4 enzyme) were widely distributed, originating from urology and gynaecology wards, emergency centre, rehabilitation centre, respiratory medicines, orthopaedic surgery, neurosurgery, general surgery, haematology, and gastroenterology, and were also isolated from outpatients. P. aeruginosa producing OXA-4 ß-lactamase was most frequently isolated (isolation rate of 21%) from the urology ward. Sixty-four per cent and 21% of the isolates from patients were isolated from urine and pus, respectively.
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Discussion |
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The OXA-4 type of ß-lactamase has been detected in E. coli and P. aeruginosa isolates, and the OXA-1 type in the Enterobacteriacea. 1 ,7 ,12 ,13 ,14 ,15 ,16 Both enzymes are plasmid-mediated. 1 ,12 However, in the DNA of the 22 isolates studied here, no plasmid DNA could be separated by the method of Kado & Lieu. 17 The 47 kb plasmid from strain FHPA1105, prepared by the PEG method (see Materials and methods), did not hybridize with the OXA-4 gene probe (Figure 5). The OXA-4 genes from these isolates were located in the band corresponding to chromosomal DNA alone (Figure 4). The OXA-4 gene was not transconjugated to the recipients. E. coli J53-2 and P. aeruginosa PAO1808 by filter mating (data not shown). These findings suggested that the OXA-4 ß-lactamase was not encoded on any plasmid. The OXA-4 gene is located on a transposon, 2 so we considered that the transposon expressing the enzyme might be located on the chromosome as well as the plasmid. Almost all of the plasmids encoding OXA-4 ß-lactamase in these P. aeruginosa isolates seemed to be spontaneously cured. Since the gene encoding the GN17262 penicillinase with pI 7.45 reported by Watanabe et al. 18was not transconjugated and the plasmid was not obtained by the two DNA isolation methods, 17,19 the penicillinase in this study may be OXA-4.
The integron reported by Collis et al. 20 possesses a drug resistance gene on its cassette gene and translocates to the plasmid depending on the sequence of the recombination site. The OXA-1 gene must be on an integron, since the 59 base element included in the integrase-dependent recombination exists downstream of the aadA gene. 11,20 The restriction endonuclease map of the EcoRI fragment of the plasmid pCR1 including the OXA-4 gene is quite similar to that of the OXA-1 gene, 2 although the nucleotide sequence was not determined. By direct sequencing based on the PCR method, the nucleotide sequence between the OXA-4 structural gene and the aadA promoter gene was consistent with that of the OXA-1 gene shown by Ouellette et al., 11 demonstrating the existence of the target sequence AAAGTT as a hot spot of the insertion (data not shown). These features revealed that the OXA-4 gene is located on an integron similar to that of the OXA-1 gene. Thus, the lack of an OXA-4 gene in the 47 kb plasmid prepared from strain FHPA1105 may result from the deletion of the integron possessing this enzyme as a cassette gene (Figure 5).
Cefclidin, but not ceftazidime, was hydrolysed by the OXA-4 and -1 enzymes tested, although both antibiotics are oxyiminocephalosporins belonging to the group of the third-generation cephalosporins. The isolates producing the OXA-4 enzyme had significant piperacillin-hydrolysing activity, and were resistant to piperacillin and ticarcillin, with MIC values of >100 mg/L. 4 Neu et al. 21 observed that, in contrast to other aminothiazolyl cephalosporins, cefclidin which is protected by a carbamoylquinuclidino group has an extremely low affinity for ß-lactamase, as in the case with the methylpyrrolidino group in cefepime. Hence, we reconfirmed that cefclidin is a novel oxyiminocephalosporin, since the affinity for OXA-4 (also OXA-1) ß-lactamase was apparently similar to that of a penem rather than that of a third-generation cephalosporin.
The 28 isolates (including the six isolates that died in storage) were widely distributed amongst the hospital wards. They constituted 15% of the total of 186 isolates collected, and 75% were O-serotype E, which was most frequently isolated amongst all O-serotypes. 4 Of the P. aeruginosa isolates collected in UK in 1993, corresponding to the collection period of this study, the three isolates producing OXA-4 ß-lactamase detected by isoelectric focusing existed at one hospital but the enzyme did not exist at the other 23 hospitals surveyed. 14 Our results suggested that non-plasmidic OXA-4 ß-lactamase in these P. aeruginosa isolates commonly colonized hospitalized patients during the collection period.
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Acknowledgments |
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Notes |
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References |
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Received 30 June 1998; returned 25 August 1998; revised 7 September 1998; accepted 9 September 1998