Prevalence of Ambler class A and D ß-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea

Seungok Lee1, Yeon-Joon Park2,*, Myungshin Kim2, Hae Kyung Lee2, Kyungja Han2, Chang Suk Kang2 and Moon Won Kang3

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


* Corresponding author. Tel: +82-2-590-1604; Fax: +82-2-592-4190; Email: yjpk{at}catholic.ac.kr

Received 1 February 2005; returned 7 March 2005; revised 11 April 2005; accepted 15 April 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Recently, resistance to extended-spectrum cephalosporins due to acquired ß-lactamases has been reported in Pseudomonas aeruginosa. The aim of this study was to investigate the prevalence of Ambler class A and D ß-lactamases and their extended-spectrum derivatives and antimicrobial susceptibilities of P. aeruginosa isolated from various parts of Korea.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is one of the bacterial species most frequently responsible for nosocomial infection and is notably resistant to many antibiotics, including ß-lactams.1 Resistance to extended-spectrum ß-lactams in P. aeruginosa is associated mostly with the overproduction of chromosomal AmpC cephalosporinase, or with non-enzymic mechanisms such as drug efflux or outer membrane impermeability.1,2 Recently, however, several class A, B and D extended-spectrum ß-lactamases (ESBLs) have been reported in P. aeruginosa.3 In contrast to the enterobacterial species in which TEM- and SHV-type enzymes are most frequent, OXA- and PSE-types are the most frequently encountered ß-lactamases in P. aeruginosa.4 Five types of class A ESBLs (PER-, VEB-, GES- and IBC-, TEM- and SHV-type) were recently reported in P. aeruginosa but they have so far been found in a limited number of geographic areas.4 OXA-type ß-lactamases, which comprise class D, have extreme sequence variation, with the identities varying from 16 to 99% between individual enzymes.5 They fall into five groups (groups I–V).6 The OXA group I includes OXA-5, 7, 10, 13 and its extended-spectrum derivatives (OXA-11, 14, 16, 17, 19). Group II includes OXA-2, 3, 15 and 20. Group III includes OXA-1, 4, 30 and 31. Group IV includes only OXA-9, and group V only LCR-1. OXA-18 does not belong to any of these groups and it has low amino acid homology. Although several surveys for class B metallo-ß-lactamases have been performed in Korea,7,8 there has been no surveillance for class A or D ß-lactamases in P. aeruginosa.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates and antimicrobial susceptibility

A total of 252 consecutive, non-duplicate P. aeruginosa isolates were collected from 12 university hospital laboratories and one commercial clinical laboratory (18–20 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 Tris–HCl, 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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Table 1. Oligonucleotides used as primers for amplification and sequencing

 
Identification of PCR products

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, Mueller–Hinton 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 Mueller–Hinton 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 0.5xTris–borate–EDTA 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 {lambda}-DNA ladder (Bio-Rad) was used as a DNA molecular weight marker.

Statistics

Statistical analysis was carried out using the {chi}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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevalence of class A and D ß-lactamases and their extended-spectrum derivatives

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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Table 2. Prevalence of Ambler class A and D ß-lactamases in 252 P. aeruginosa isolates

 
RFLP analysis was performed on the 760 bp OXA-10-like (OXA group I) amplification products from 34 isolates. All were cleaved into two fragments (312 and 448 bp) by PvuII, and into three fragments (198, 240 and 332 bp) by HaeIII, indicating that the alleles were either blaOXA-10 or blaOXA-17; digestion with HhaI confirmed that 33 isolates contained blaOXA-10 (fragments of 120 and 640 bp) and one isolate with three fragments (120, 66 and 574 bp) was blaOXA-17, an OXA-10-derived ESBL. They showed 100% identity with blaOXA-10 (U37105) and blaOXA-17 (AF060206), respectively by sequencing analysis. Of the 16 OXA-1-positive (OXA group III) PCR products, 11 were OXA-4 (identity with AY162283) and five were OXA-30 (identity with AF255921). Similarly, OXA-2 and PSE-1 PCR products showed 100% identity with blaOXA-2 (X03037) and blaPSE-1 (M69058), respectively. IEF analysis showed pI values that were consistent with the gene identified: pI 6.1 for blaOXA-10 and blaOXA-17; pI 7.7 for blaOXA-2; pI 7.5 for blaOXA-4; pI 7.3 for blaOXA-30; and pI 5.7 for blaPSE-1.21,22 All isolates, including the OXA-17 producer, showed a negative reaction for DDST, which coincides with the PCR results. The ß-lactamases produced by P. aeruginosa isolates varied between provinces (Figure 1). In the central districts of Korea (Seoul, Kyungki, Kangwon and Choongchung area), OXA-10 was most common, but in the southern part (Chulla and Kyunsang), OXA-30 and OXA-4 were more common. However, in the majority of cases, the PFGE profiles of isolates harbouring the same enzyme were different; exceptions included a few clonally-related isolates containing blaPSE-1 and blaOXA-10 in Seoul, blaOXA-10 in Kangwon and Choongchung, and blaOXA-4 and blaPSE-1 in Chulla (data not shown).



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Figure 1. Distribution of the types of 72 class A and D ß-lactamases detected in 64 P. aeruginosa isolates from cities and provinces of Korea. The number of ß-lactamases detected is shown at the top of each column. Shading (working up first Seoul column for order): mid-grey pattern, OXA-10; diagonal brick pattern, OXA-2; dark grey pattern, OXA-4; diagonal pattern, OXA-30; light grey pattern, PSE-1; dashed pattern (in Kyungki column), OXA-17.

 
Comparative analysis of antimicrobial resistance between PSE or OXA type ß-lactamase producers and non-producers

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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Table 3. Antimicrobial susceptibility profiles of 252 P. aeruginosa isolates

 

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Table 4. Comparison of the antimicrobial resistance (%) between the class A or D ß-lactamase producers and non-producers

 
The sensitivity and specificity of antibiotic resistance profiles for detecting PSE- or OXA-type ß-lactamase were compared and are summarized in Table 5; combined resistance to ticarcillin and tobramycin or gentamicin revealed excellent sensitivity and specificity (sensitivity >90%, specificity >90%).


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Table 5. Sensitivity and specificity of ticarcillin or piperacillin resistance and their resistance combined with aminoglycosides or ciprofloxacin for detecting PSE or OXA type ß-lactamase producers

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Class D OXA ß-lactamases were more frequently detected than class A in P. aeruginosa from Korea (21.0% versus 6.3%), which contrasts with data from Europe (31.3% versus 64.9%).6 In Korea, OXA-10 was the most prevalent enzyme (13.5%), followed by OXA-4 (4.4%), OXA-2 (2.3%) and OXA-30 (2.0%).

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.


    Acknowledgements
 
We thank the Korean Nationwide Surveillance of Antimicrobial Resistance Group for sending P. aeruginosa clinical isolates. We thank Professor P. Nordmann (Service de Bactériologie-Virologie, Hôpital de Bicêtre, France), K. Lee (Yonsei University College of Medicine, Korea) and H. Pai (Hanyang University College of Medicine, Korea) for giving professional advice and for sending ß-lactamase-positive control isolates. We also thank J. J. Park and S. Y. Park in our laboratory for excellent technical assistance. This work was supported by a Korea Research Foundation grant (KRF-2003-041-E00202).


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 Introduction
 Materials and methods
 Results
 Discussion
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