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Koo, Kwon, Cho, and Sung: Genetic Basis of Multidrug-resistant Acinetobacter baumannii Clinical Isolates from Three University Hospitals in Chungcheong Province, Korea
Original article

Korean J Leg Med 2010;30(5):498-506.

Published online: 14 January 2010

DOI: https://doi.org/10.3343/kjlm.2010.30.5.498

Genetic Basis of Multidrug-resistant Acinetobacter baumannii Clinical Isolates from Three University Hospitals in Chungcheong Province, Korea

Sun Hoe Koo, M.D.1, Kye Chul Kwon, M.D.1, Hye Hyun Cho, B.S.1, Ji Youn Sung, Ph.D.2

1Department of Laboratory Medicine, College of Medicine, Chungnam National University, Daejeon, Korea

2Department of Biomedical Laboratory Science, Far East University, Eumseong, Korea

Corresponding author: Ji Youn Sung, Ph.D. Department of Biomedical Laboratory Science, Far East University, Wangjang-ri, Gamgok-myeon, Eumseong 369-700, Korea Tel: +82-43-879-3668, Fax: +82-43-879-3730 E-mail: azaza72@naver.com

∗ This study was financially supported by the Chungnam National University Research Fund in 2009.

      Revised 15 July 201012 August 2010       Accepted 13 August 2010

Copyright © 2010 Korean Society for Legal Medicine

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:

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii as an important opportunistic pathogen has given rise to significant therapeutic challenges in the treatment of nosocomial infections. In the present study, we assess the antibiotic resistance mechanisms of MDR A. baumannii strains by estimating the prevalence of antibiotic resistance determinants, including integrons, β-lactamases, str genes, and gyrA and parC mutations.

Methods:

Thirty-five MDR A. baumannii clinical isolates were collected from 3 Korean university hospitals over a 2-yr period. A. baumannii was confirmed by rpoB gene analysis. For each isolate, the minimal inhibitory concentrations (MICs) of 9 antibiotics were determined by the agar dilution method. PCR and DNA sequencing were used to identify the genes that potentially contribute to each resistance phenotype.

Results:

Of the 35 MDR A. baumannii isolates examined, 7 antibiotic resistance gene determinants were detected. These resistance gene determinants included the gene blaOXA-23, with an upstream element ISAba1 to promote increased gene expression and subsequent resistance to carbapenems, in 8 isolates (22.9%); aacA4, located within class 1 integrons, in 7 isolates (20.0%); and fluoroquinolone resistance conferred by gyrA and parC sense mutations in 31 isolates.

Conclusions:

Of the 35 MDR A. baumannii isolates, 26 (74.3%) from both outbreak and sporadic cases possessed at least 4 of the 7 antibiotic resistance gene determinants that give rise to the MDR phenotype. The co-occurrence of several resistance determinants may present a significant threat.

Keywords: MDR A. baumannii, Antibiotic resistance gene determinants, ISAba1

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Fig. 1.
Schematic representation of the gene cassette structure located in the class 1 integron isolated from multidrug-resistant Acinetobacter baumannii. The horizontal arrows indicate the translation orientation of the genes.
kjlm-30-498f1.tif
Fig. 2.
Repetitive element sequence-based (REP)-PCR patterns of genomic DNA from 35 multidrug resistant A. baumannii. Lane M contains a 1 kb DNA size marker.
kjlm-30-498f2.tif
Table 1.
Primers used in this study
Primer Target gene/region Sequence (5′→3′) Reference
PW166 ISAba1 CCTATCAGGGTTCTGCCTTCT 15
PW200 strA ATGATGTCTAACAGCAAACTG 15
PW201   TCAACCCCAAGTAAGAGG  
PW202 strB ATGGGGTTGATGTTCATGCCGC 15
PW203   CTAGTATGACGTCTGTCGCAC  
TEM F blaTEM-1 and derivative ATGAGTATTCAACATTTCCGT 5
TEM R   TTACCAATGCTTAATCAGTGA  
SHV F blaSHV-1 and derivative CCGGGTTATTCTTATTTGTCGCT 5
SHV R   TAGCGTTGCCAGTGCTCG  
CTX-M F blaCTX-M- 1, 2, 9 group GATTGACCGTATTGGGAGTTT 5
CTX-M R   CGGCTGGGTAAAATAGGTCA  
PER F blaPER-1 GTTAATTTGGGCTTAGGGCAGA 5
PER R   CAGCGCAATCCCCACTGT  
VEB F blaVEB-1 CGACTTCCATTTCCCGATGC 5
VEB R   GGACTCTGCAACAAATACGC  
GES F blaGES-1, −2, −3, −4, blaIBC-1 GTTAGACGGGCGTACAAAGATAAT 5
GES R   TGTCCGTGCTCAGGATGAGT  
PSE F blaPSE-1 AATGGCAATCAGCGCTTC 5
PSE R   GCGCGACTGTGATGTATA  
IMP F blaIMP CATGGTTTGGTGGTTCTTGT 5
IMP R   ATAATTTGGCGGACTTTGGC  
VIM F blaVIM ATTGGTCTATTTGACCGCGTC 5
VIM R   TGCTACTCAACGACTGAGCG  
OXA-23-like F blaOXA-23-like CTTGCTATGTGGTTGCTTCTC 15
OXA-23-like R   ATCCATTGCCCAACCAGTC  
OXA-24-like F blaOXA-24-like GTACTAATCAAAGTTGTGAA 5
OXA-24-like R   TTCCCCTAACATGAATTTGT  
OXA-51-like F blaOXA-51-like ATGAACATTAAAGCACTC 16
OXA-51-like R   CTATAAAATACCTAATTGTTC  
OXA-58-like F blaOXA-58-like CGATCAGAATGTTCAAGCGC 5
OXA-58-like R   ACGATTCTCCCCTCTGCGC  
GyrA F gyrA AAATCTGCCCGTGTCGTTGGT 17
GyrA R   GCCATACCTACGGCGATACC  
ParC F parC AAGCCCGTACAGCGCCGTATT- 18
ParC R   AAAGTTATCTTGCCATTCGCT  
Table 2.
MICs of the 9 antibiotics for the 35 Acinetobacter baumannii isolates as determined by agar dilution
Isolates MIC (mg/L)
AMK GEN STR AMP CAZ CTX FEP IPM CIP
A4 64 <16 32 128 128 128 64 16 4
A14 64 >128 >512 >512 512 >512 64 64 64
A15 <2 <16 64 128 128 128 64 16 4
A16 16 >128 >512 >512 >512 >512 256 16 >64
A20 <2 >128 >512 >512 >512 >512 256 64 >64
A22 6 >128 >512 >512 128 512 32 8 >64
A23 64 >128 256 >512 128 256 64 16 >64
A31 4 >128 >512 >512 512 >512 32 16 >64
A32 4 >128 >512 >512 256 512 32 16 >64
A34 4 >128 >512 >512 256 512 32 16 >64
A36 4 >128 >512 >512 512 512 32 16 >64
B1 64 >128 >512 >512 512 >512 64 128 >64
B3 64 >128 >512 >512 >512 >512 256 16 >64
B4 64 >128 >512 >512 512 512 64 64 >64
B6 64 >128 >512 >512 >512 >512 256 64 >64
B9 64 >128 >512 >512 >512 >512 256 64 >64
B13 64 >128 >512 >512 512 >512 64 64 >64
B21 64 >128 >512 >512 128 512 64 16 >64
B22 64 >128 >512 >512 128 512 64 16 >64
B23 64 >128 >512 >512 128 512 64 16 >64
B24 64 >128 >512 >512 >512 >512 >512 16 >64
B25 64 >128 >512 >512 >512 512 512 16 >64
B27 64 >128 >512 >512 128 512 64 16 >64
B28 64 >128 >512 >512 128 512 64 16 >64
B29 64 >128 >512 >512 128 512 64 16 >64
B30 64 >128 >512 >512 128 512 64 16 >64
B33 64 >128 >512 >512 128 256 64 16 >64
B36 64 >128 >512 >512 128 512 64 16 >64
B37 64 >128 >512 >512 128 512 64 16 >64
C1 64 64 64 >512 >512 256 64 64 4
C4 64 64 64 >512 >512 128 64 64 64
C6 64 >128 >512 >512 128 512 64 64 >64
C8 64 >128 >512 >512 >512 >512 512 64 >64
C11 64 >128 >512 >512 512 512 64 64 >64
C25 4 <16 32 128 128 128 64 16 4

Abbreviations: MIC, minimum inhibitory concentration; AMK, amikacin; GEN, gentamicin; STR, streptomycin; AMP, ampicillin; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; IPM, imipenem; CIP, ciprofloxacin.

Table 3.
Profiles of antibiotic resistance determinants in multidrug-resistant Acinetobacter baumannii Isolates
Isolates Antibiotic resistance determinants in MDR A. baumannii
strB ISAba1/blaoxa-23 ISAba1/blaoxa-51-like blaIMP-1 gyrA mutation parC mutation Integron
A4 - - - - - - -
A14 + + - - + + Type 2
A15 - - - - - - -
A16 + - + - + + -
A20 + + - - + + -
A22 + - + - + + -
A23 - - - - + + -
A31 + - + - + + -
A32 + - + - + + -
A34 + - + - + + -
A36 + - + - + + -
B1 + + - - + + -
B3 + - - - + + -
B4 + + - - + + -
B6 + + + - + + Type 2
B9 + + + - + + Type 2
B13 + + - - + + Type 2
B21 + - + - + + -
B22 + - + - + + -
B23 + - + - + + -
B24 + - - - + + -
B25 + - + - + + -
B27 + - + - + + -
B28 + - + - + + -
B29 + - + - + + -
B30 + - + - + + -
B33 - - + - + + -
B36 + - + - + + -
B37 - - + - + + -
C1 - - - + - - Type 1
C4 - - - + + + Type 1
C6 + - + - + + -
C8 + - + - + + -
C11 + + - - + + -
C25 - - - + - - Type 1
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