1 Seoul Medical Science Institute, Seoul Clinical Laboratories, Seoul, Korea; 2 Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Kangnam St Mary's Hospital, 505 Banpo-dong, Seocho-ku, Seoul, 137-701, Korea; 3 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea
Received 1 February 2005; returned 7 March 2005; revised 11 April 2005; accepted 15 April 2005
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Abstract |
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Methods: A total of 252 consecutive, non-duplicate isolates of P. aeruginosa were studied for the presence of class A or D ß-lactamase. Antibiotic susceptibility tests and PCR amplification of genes encoding class A (blaPSE-1, blaPER-1, blaVEB-1, blaTEM, blaSHV, blaCTX-M and blaGES-1) and class D ß-lactamases (blaOXA-groupI, blaOXA-groupII and blaOXA-groupIII) were performed. For PCR-positive isolates, isoelectric focusing (IEF) analysis, sequencing and pulsed-field gel electrophoresis (PFGE) were performed.
Results: In 64 (25.4%) isolates, structural genes for PSE-1 (6.3%), OXA-10 (13.1%), OXA-4 (4.3%), OXA-30 (2.0%), OXA-2 (2.3%) and OXA-17 (0.4%) were found; their distribution varied between provinces. None harboured blaPER-1, blaVEB-1, blaTEM, blaSHV, blaCTX-M and blaGES-1. The cross-class resistance rates to other antibiotics was significantly higher in class A and D ß-lactamase producers than in non-producers (P < 0.001 for aminoglycosides, ciprofloxacin and meropenem).
Conclusions: OXA-type ß-lactamases are widespread, but their extended-spectrum derivatives are rare among P. aeruginosa in Korea. To our knowledge, this is the first report of OXA-17, an extended-spectrum derivative of OXA-10, outside the Middle East. In addition, combined resistance to ticarcillin and aminoglycosides was a useful indicator for P. aeruginosa producing PSE- or OXA-type ß-lactamases in this study.
Keywords: P. aeruginosa , ESBLs , extended-spectrum ß-lactamases , OXA-17
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Introduction |
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This study was performed to investigate the nationwide prevalence of Ambler class A and D ß-lactamases and their extended-spectrum derivatives in clinical isolates of P. aeruginosa.
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Materials and methods |
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A total of 252 consecutive, non-duplicate P. aeruginosa isolates were collected from 12 university hospital laboratories and one commercial clinical laboratory (1820 isolates per centre) in a nationwide distribution (Seoul, 7; Kyungki, 2; Kangwon, 1; Choongchung, 1; Chulla, 1; Kyungsang, 1) between April and June 2002. Isolates were from sputum (45%), drainage (14%), urine (12%), wound (7%) and others from patients in inpatient departments (48%), intensive care units (31%) or outpatient departments (21%). The isolates were stored at 76°C in 20% skimmed milk until used in this study.
Antimicrobial susceptibility tests were performed by the disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines.9 The antimicrobial discs used were piperacillin, piperacillin/tazobactam, ticarcillin, ticarcillin/clavulanic acid, ceftazidime, aztreonam, cefepime, gentamicin, amikacin, tobramycin, netilmicin, ciprofloxacin, imipenem and meropenem (BBL, Cockeysville, MD, USA). Antimicrobial susceptibility profiles were compared for class A and D ß-lactamase producers and non-producers.
PCR amplification of ß-lactamase genes
The total DNA from P. aeruginosa isolates was extracted by boiling. PCR was carried out with 2 µL of the template DNA, 0.5 µM of each primer, 10 mM TrisHCl, 100 µM dNTP and 1.5 U of Taq DNA polymerase (Takara Shuzo, Shiga, Japan) in a total volume of 50 µL. Specific primers for detection of ß-lactamases (blaPER-1, blaVEB-1, blaOXA-groupI, blaOXA-groupII, blaOXA-groupIII, blaPSE, blaTEM, blaSHV, blaCTX-M, blaGES-1) were used (Table 1).1017 Control strains harbouring blaOXA-10, blaVEB-1, blaOXA-2, blaPSE-1, blaGES-1 (all kindly provided by P. Nordmann, Service de Bactériologie-Virologie, Hôpital de Bicêtre, France), blaPER-1 (from H. Pai, Hanyang University College of Medicine, Korea), blaCTX-M-12 (from K. Lee, Yonsei University College of Medicine, Korea), blaTEM-1, blaSHV-1 and blaOXA-1 (isolated in our laboratory and confirmed by sequencing) were used. Each PCR was performed according to previously reported conditions in a Gene Amp PCR system 9600 thermocycler (Perkin-Elmer, Branchburg, NJ, USA) or a PTC-100 Thermal Cycler (MJ Research, Inc., Watertown, MA, USA).
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OXA-10 PCR products were identified by RFLP analysis with PvuII (discriminates OXA-10/11/14/16/17 from OXA-7/13), HaeIII (discriminates OXA-10/17 from OXA-11/14/16) and HhaI (discriminates OXA-17 from OXA-10).6,18 Selected PCR products were purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) and sequenced on a 3730 DNA analyser (Applied Biosystems, Foster, CA, USA). The nucleotide and deduced protein sequences were analysed with software available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).
Double disc synergy test (DDST)
As ESBLs can be obscured by the chromosomal AmpC cephalosporinase in P. aeruginosa, MuellerHinton agar containing 250 mg/L of cloxacillin (Sigma Chemical Inc., MO, USA) was prepared.10 After overnight culture, test isolates were suspended to the turbidity of a 0.5 McFarland standard and used to inoculate a MuellerHinton agar plate. After drying, discs containing ceftazidime, aztreonam and cefepime were placed 2 cm from a disc containing amoxicillin/clavulanic acid (BBL).19 After 18 h of incubation, the presence of enlarged inhibition zones was interpreted as being DDST-positive, i.e. when there was clear augmentation of the inhibition zone for any of the indicator antibiotics by amoxicillin/clavulanic acid.
Isoelectric focusing (IEF) analysis of ß-lactamases
Crude ß-lactamase preparations, derived from the sonicated bacterial cultures of the Pseudomonas isolates, were assessed for ß-lactamase pIs and inhibitor profiles by IEF, which was performed at room temperature on a Bio-Rad mini isoelectric focusing III (Bio-Rad, Richmond, CA, USA). The enzymes were visualized with 0.5 mM nitrocefin (BBL), and pIs were estimated by comparison with TEM-1, TEM-10, SHV-1, SHV-5 and CMY-1 standards.
Pulsed-field gel electrophoresis (PFGE) analysis
PFGE was performed according to the manufacturer's protocol (Bio-Rad). Briefly, whole-cell DNA of P. aeruginosa isolates harbouring class A or D ß-lactamases was digested with SpeI restriction enzyme for 4 h at 50°C.20
Electrophoresis was performed with a CHEF DRII (Bio-Rad) through a 1% agarose gel in 0.5xTrisborateEDTA buffer at 14°C, voltage of 6 V/cm and switch angle of 60°, using a pulse time ranging from 5 to 50 s for 24 h. A bacteriophage -DNA ladder (Bio-Rad) was used as a DNA molecular weight marker.
Statistics
Statistical analysis was carried out using the 2 test with the SPSS program (SPSS 10.0 for Windows, SPSS Inc., Chicago, IL, USA). P values of <0.05 were considered to be significant.
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Results |
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Of the 252 isolates, 53 (21.0%) isolates harboured OXA-type and 16 (6.3%) harboured PSE-type enzyme (Table 2). Of the OXA ß-lactamases, OXA-10 was most prevalent, followed by OXA-4, OXA-2, OXA-30 and OXA-17. Six isolates (2.4%) harboured two different ß-lactamases and one harboured three enzymes (Table 2). PER-1, VEB-1, TEM, SHV, CTX-M and GES-1 enzymes were not detected.
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The antibiotic susceptibility profiles of the 252 isolates are summarized in Table 3. Over 70% of isolates were susceptible to imipenem, meropenem, ceftazidime, cefepime, amikacin, netilmicin, tobramycin and piperacillin/tazobactam. The resistance rates to most antibiotics were significantly higher in PSE- or OXA-producers except ceftazidime and imipenem (Table 4).
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Discussion |
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The OXA-10 derivatives (group I) confer greater resistance to ceftazidime than to cefepime.13 In contrast, OXA-1 and its group III derivatives (OXA-4, OXA-30 and OXA-31) characteristically show decreased susceptibility to cefepime, but remain susceptible to ceftazidime.13,23 In this study, among the seven isolates that showed resistance to cefepime and susceptibility to ceftazidime, two of them harboured blaOXA-30 and two (28.6%) harboured blaOXA-4.
One Korean isolate (AJ-07) harboured OXA-17, an extended-spectrum derivative of OXA-10. To our knowledge, this is the first report of OXA-17 outside the Middle East. Among the extended spectrum derivatives of OXA-10, OXA-11, -14, and -16 confer a high level of resistance to ceftazidime (MIC > 128 mg/L) and have a Gly-157 Asp substitution, which may be critical to ceftazidime resistance. In contrast, OXA-17 has an Asn-73
Ser substitution, which has minimal effects on the MIC of ceftazidime.24
The ceftazidime MIC for isolate AJ-07 was 8 mg/L, which is considered susceptible according to interpretative standards of NCCLS.25
Although five types of class A ESBLs (PER-, VEB-, GES- and IBC-, TEM- and SHV-type) were recently reported in P. aeruginosa, none of these was observed in this study. It might be related to the lower ceftazidime resistance rate (16.7%) compared with that in Turkey (28%), where PER-1 ESBL was a major problem.26 In Korea, PER-1 is widespread in Acinetobacter spp., but has not been reported in Pseudomonas spp.27
In P. aeruginosa, ticarcillin has been used as a marker of multiresistance; resistance is caused by production of acquired ß-lactamase, overproduction of constitutive cephalosporinase or non-enzymic mechanisms, such as hyperactive efflux or reduced permeability. Production of PES-1 confers very high resistance to ticarcillin.1 In this study, of 125 isolates resistant to ticarcillin, 14 isolates produced PSE-1 and 47 produced OXA-type ß-lactamase. The cross-class resistance to aminoglycosides and quinolone was significantly higher in class A or D ß-lactamase producing P. aeruginosa in this study. This can pose the substantial risk of treatment failures as in ESBL producers.28,29 To make things more serious, in vivo selection of an extended-spectrum derivative from a restricted-spectrum oxacillinase producing P. aeruginosa was reported during a ceftazidime-containing treatment.30 In addition, of the various ß-lactamase genes, blaOXA-2- and blaPSE-1-related genes were the only ones detected within phage particles from sewage samples and this finding suggests that phages potentially contribute to the spread of these resistance genes.31,32
In conclusion, OXA-type ß-lactamases of diverse clonal origins are widespread in Korea, but not those derivatives with extended activity spectra. Taking into account the threat of cross-resistance, potential for spread by phages, and in vivo selection of ESBL, clinical efforts for the early recognition of acquired ß-lactamase-producing strains and rigorous infection control should be emphasized. Although it needs further investigation, the combined resistance to ticarcillin and tobramycin or gentamicin could be a potential marker for production of PSE- or OXA-type ß-lactamase in P. aeruginosa, as noted in this study.
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Acknowledgements |
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