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Chung, Sung, Kwon, Park, Ko, Shin, Song, and Koo: Characteristics of Acquired β-lactamase Gene in Clinical Isolates of Multidrug-resistant Pseudomonas aeruginosa
Original Article

Korean J Clin Microbiol 2008;11(2):98-106.

Published online: 20 October 2008

DOI: https://doi.org/10.5145/kjcm.2008.11.2.98

Characteristics of Acquired β-lactamase Gene in Clinical Isolates of Multidrug-resistant Pseudomonas aeruginosa

Sun Yang Chung1, Ji Youn Sung2, Kye Chul Kwon2, Jong Woo Park2, Chi Seon Ko2, So Youn Shin2, Jeong Hoon Song2, Sun Hoe Koo2,

1Health Promotion Center, Samsung Medical Center, Seoul, Korea

2Department of Laboratory Medicine, Chungnam National University College of Medicine, Daejeon, Korea

Correspondence: Sun Hoe Koo, Department of Laboratory Medicine, Chungnam National University Hospital, 640, Daesa-dong, Jung-gu, Daejeon, Korea. (Tel) 82-42-280-7798, (Fax) 82-42-257-5365, (E-mail) shkoo@cnu.ac.kr

Received 31 January 2008       Accepted 20 August 2008

Copyright © 2008 Korean Journal of Clinical Microbiology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Recently, there have been reports of in-fections with multidrug-resistant Pseudomonas aeruginosa. To determine the mechanism of the resistance, we investigated the prevalence of Ambler class A and D β-lactamases, their extended-spectrum derivatives, and class B and D carbapenemase in mul-tidrug-resistant P. aeruginosa isolates.

Methods

During the period of March 2006 to May 2007, clinical isolates of multidrug-resistant P. aeruginosa were collected from patients in Chungnam National University Hospital, Daejeon, Korea. Inhibitor-potentiated disk diffusion tests were used for the screening of metallo-β-lactamase (MBL) production. PCR and DNA sequencing were conducted for the detection of β-lactamase genes. We also employed the enterobacterial repetitive intergenic consensus (ERIC)-PCR method for an epidemiologic study.

Results

A total of 37 consecutive, non-duplicate, multidrug-resistant P. aeruginosa were isolated. Twentynine of 37 isolates harbored blaOXA-10 (56.8%), blaOXA-2 (18.9%), and blaOXA-1 (5.4%). Only one iso-late produced IMP-1, and it also harbored blaOXA-1. None harbored Ambler class A β-lactamase or class D carbapenemase. The strains producing OXA type β-lactamases showed a significantly higher resistance to aminoglycoside compared to non-producers. The ERIC-PCR pattern of the 19 OXA-10 producing strains indicated that the isolates were closely related in terms of clonality.

Conclusion

OXA type β-lactamases are the most prevalent among the acquired β-lactamases produced by multidrug-resistant P. aeruginosa isolated at a university hospital in Chungcheong Province. Besides β-lactam antibiotics, the strains harboring OXA type β-lactamase showed a significantly higher resistance to aminoglycoside and qunolone.

Keywords: Multidrug resistance, Pseudomonas aeruginosa, OXA type β-lactamase, Metallo-β-lactamase (MBL)

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Fig. 1.
ERIC-PCR patterns of genomic DNA from clinical isolates of multi-drug resistant Pseudomonas aeruginosa harboring blaOXA-10. Lane M is 1 kb DNA size marker. Nineteen strains of OXA-10 β-lactamase producing clinical isolates show A or B pattern.
kjcm-11-98f1.tif
Table 1.
Oligonucleotides used as primers for amplification and sequencing
Enzyme Class Primer pairs Target Sequence (5'-3') Amplicon size (bp) Reference
Class A TEM F TEM R blaTEM and derivative ATGAGTATTCAACATTTCCGT TTACCAATGCTTAATCAGTGA 861 24
SHV F SHV R blaSHV and derivative TAGCGTTGCCAGTGCTCG CCGGGTTATTCTTATTTGTCGCT 831 24
PER F PER R blaPER CAGCGCAATCCCCACTGT GTTAATTTGGGCTTAGGGCAGA 855 26
VEB F VEB R blaVEB CGACTTCCATTTCCCGATGC GGACTCTGCAACAAATACGC 650 25
GES F GES R blaGES, blaIBC TGTCCGTGCTCAGGATGAGT GTTAGACGGGCGTACAAAGATAAT 903 24
Class B IMP F IMP R blaIMP CATGGTTTGGTGGTTCTTGT ATAATTTGGCGGACTTTGGC 488 27
VIM F VIM R blaVIM ATTGGTCTATTTGACCGCGTC TGCTACTCAACGACTGAGCG 780 27
SIM F SIM R blaSIM GTACAAGGGATTCGGCATCG TGGCCTGTTCCCATGTGAG 569 31
SPM F SPM R blaSPM CTAAATCGAGAGCCCTGCTTG CCTTTTCCGCGACCTTGATC 798 31
GIM F GIM R blaGIM TCAATTAGCTCTTGGGCTGAC CGGAACGACCATTTGAATGG 72 31
Class D OXA-1F OXA-1R blaOXA group III AGCCGTTAAAATTAAGCCC CTTGATTGAAGGGTTGGGCG 908 28
OXA-2F OXA-2R blaOXA group II GCCAAAGGCACGATAGTTGT GCGTCCGAGTTGACTGCCGG 700 29
OXA-10F OXA-10R blaOXA group I TCTTTCGAGTACGGCATTAGC CCAATGATGCCCTCACTTTCC 760 25
OXA-23F OXA-23R blaOXA-23, 27, 49 GATGTGTCATAGTATTCGTCG TCACAACAACTAAAAGCACTG 1,058 27
OXA-24F OXA-24R blaOXA-24, 25, 26, 40, 72 GTACTAATCAAA GTTGTGAA TTCCCCTAACATGAATTTGT 825 27
OXA-58F OXA-58R blaOXA-58 CGATCAGAATGTTCAAGCGC ACGATTCTCCCCTCTGCGC 528 30

Abbreviations: F, forward; R, reverse.

Table 2.
Antibiotic susceptability profiles and β-lactamases of multi-drug resistant Pseudomonas aeruginosa
Isolates Antibiotic susceptibilities β-lactamase
AMK GEN NET TOB ATM CAZ FEP IPM MEM PIP TZP TIC TIM CIP β-lactamase
P2 R R R R R I I R R S S R R R  
P3 R R R S R R R R R S R R R R  
P4 R R R R S S R R R S S R S R OXA-10
P5 R R R R R I R R R R R R R R OXA-10
P7 I R R S R R R R R R R R R R  
P15 R R R R R R R R R R R R R R OXA-2
P17 R R R R R R I R R R R R S R OXA-2,
P18 S R R R R S S R R R R R R R OXA-10
P21 R R R R R R S R R S S R R R  
P23 R R R R I R R R R R R R R R  
P25 R R R R R R R R R S S R S R OXA-1, IMP
P26 R R R R R R R R R R R R R R OXA-10
P29 R R R R R R R R R R R R R R OXA-2
P31 R R R R R R R R R R S R R R OXA-2
P34 S S S S R R R R R R R R R S OXA-2
P35 R R R R R I I R R R S R R R  
P40 R R R R R R R R R R R R R R OXA-10
P41 R R R R R I R R R R S R S S OXA-2
A2 R R R R I R R S S R R R R R  
A3 R R R R I R R I I S S R R R OXA-10
A8 R R R R R R R I R R R R R R OXA-10
A9 R R S R I R R I I S S R S R OXA-10
A10 R R R R R I I R R R S R R R OXA-10
A11 R R R R I R R I I S S R R R OXA-10
A13 R R R S I I I R R S S S R R OXA-10
A14 R R R R I R R I I S S R R R  
A15 R R R R I R R R I S S R R R OXA-10
A16 R R R R R R R I R R R R R R OXA-10
A18 R R R R I R R I I S S R R R OXA-10
A19 R R R R R R R R R R R R R R OXA-10
A26 R R R S R R R R R S R R R R OXA-2
A32 R R R R I R R I I S S R R R  
A36 R R R R I R I R R R R R R R OXA-10
A41 R R R R I R R I I S S R R R OXA-10
A42 R R R R I R R I I S S R R R OXA-10
A43 R R R R I R R S S R R R R R OXA-10
A50 R R S R S S R R I S S R S R OXA-1

Abbreviations: AMK, amikacin; GEN, gentamicin; NET, netilmicin; TOB, tobramycin; ATM, aztreonam; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; MEM meropenem; PIP, piperacillin; TZP, piperacillin-tazobactam; TIC, ticarcillin; TIM, ticarcillin-clavulanic acid; CIP, ciprofloxacin.

Table 3.
Prevalence of Ambler class A, B, and D β-lactamases in multi-drug resistant Pseudomonas aeruginosa
Class Type of β-lactamases No.(%) of isolates
Class B IMP-1 1 (2.7)∗7
Class D OXA-1 2 (5.4)∗
  OXA-2 7 7 (18.9)†
  OXA-10 20 (54.1)†
Combined IMP and OXA-1 1 (2.7)∗
Combined OXA-2 and OXA10 7 1 (2.7)†7
Not detected   9 (24.3)

∗ P23 strain: IMP-1 was combined with OXA-1;

† P17 strain: OXA-2 was combined with OXA-10.

Table 4.
Comparison of the antimicrobial resistance (%) between the class D β-lactamase producers and non-producers
Antimicrobialagents Class D β-lactamase producers (N=28) Class D β-lactamase non-producers (N=9)
MIC50 MIC90 Resistant (%) MIC50 MIC90 Resistant (%)
AMK ≥64 ≥64 100 ≥64 ≥64 66.7
GEN ≥16 ≥16 100 ≥16 ≥16 88.9
NET ≥32 ≥32 92.9 ≥32 ≥32 88.9
TOB ≥16 ≥16 100 ≤4 ≥16 44.4
ATM 16 ≥32 46.4 ≥32 ≥32 88.9
CAZ ≥32 ≥32 82.1 ≥32 ≥32 55.6
FEP ≥32 ≥32 82.1 ≥32 ≥32 55.6
IPM ≥16 ≥16 57.1 ≥16 ≥16 100
MEM ≥16 ≥16 57.1 ≥16 ≥16 100
PIP ≥128 ≥128 57.1 ≤64 ≥128 44.4
TIC ≥128 ≥128 100 ≥128 ≥128 88.9
CIP ≥4 ≥4 92.9 ≥4 ≥4 77.8

Abbreviations: See Table 2.

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