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Song, Lee, Koh, Ha, Jeong, Bae, and Jeong: Antibiotic Resistance Mechanisms of Escherichia coli Isolates from Urinary Specimens
Original Article

Korean J Leg Med 2009;29(1):17-24.

Published online: 14 February 2009

DOI: https://doi.org/10.3343/kjlm.2009.29.1.17

Antibiotic Resistance Mechanisms of Escherichia coli Isolates from Urinary Specimens

Sungwook Song, M.D.1, Eun Young Lee, M.D.1, Eun-Mi Koh, M.D.1, Ho Sung Ha, M.D.2, Ho Joong Jeong, M.D.2, Il Kwon Bae, Ph.D.3, Seok Hoon Jeong, M.D.1,

1Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea

2Department of Physical Medicine and Rehabilitation, Kosin University College of Medicine, Busan, Korea

3Research Institute for Antimicrobial Resistance, Kosin University College of Medicine, Busan, Korea

Corresponding author: Seok Hoon Jeong, M.D. Department of Laboratory Medicine, Yonsei University College of Medicine, 250 Seongsan-ro, Sedaemun-gu, Seoul 120-752, Korea Tel: +82-2-2228-2448, Fax: +82-2-313-0956 E-mail: kscpjsh@yuhs.ac

∗This work was supported by Yonsei University Research Fund of 2008.

Received 28 July 20084 October 2008       Accepted 6 October 2008

Copyright © 2009 by the 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

This study was designed to characterize urinary isolates of Escherichia coli that produce extended-spectrum β-lactamases (ESBLs) and to determine the prevalence of other antimicrobial resistance genes.

Methods

A total of 264 non-duplicate clinical isolates of E. coli were recovered from urine specimens in a tertiary-care hospital in Busan in 2005. Antimicrobial susceptibility was determined by disk diffusion and agar dilution methods, ESBL production was confirmed using the double-disk synergy (DDS) test, and antimicrobial resistance genes were detected by direct sequencing of PCR amplification products. E. coli isolates were classified into four phylogenetic biotypes according to the presence of chuA, yjaA, and TSPE4.

Results

DDS testing detected ESBLs in 27 (10.2%) of the 264 isolates. The most common type of ESBL was CTX-M-15 (N=14), followed by CTX-M-3 (N=8) and CTX-M-14 (N=6). All of the ESBL-producing isolates were resistant to ciprofloxacin. PCR experiments detected genes encoding DHA-1 and CMY-10 AmpC β-lactamases in one and two isolates, respectively. Also isolated were 5 isolates harboring 16S rRNA methylases, 2 isolates harboring Qnr, and 19 isolates harboring AAC(6′)-Ib-cr. Most ESBL-producing isolates clustered within phylogenetic groups B2 (N=14) and D (N=7).

Conclusion

CTX-M enzymes were the dominant type of ESBLs in urinary isolates of E. coli, and ESBL-producing isolates frequently contained other antimicrobial resistance genes. More than half of the urinary E. coli isolates harboring CTX-M enzymes were within the phylogenetic group B2.

Keywords: Escherichia coli, Extended-spectrum β-lactamases (ESBL), Phylogenetic group

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Table 1.
Primers used in this study
PCR target Primer name Primer sequence Reference
blaCTX-M (CTX-M-1 cluster) CTX-M-1F 5′-GGACGTACAGCAAAAACTTGC-3′  
  CTX-M-1R 5′-CGGTTCGCTTTCACTTTTCTT-3′  
blaCTX-M (CTX-M-2 cluster) CTX-M-2F 5′-CGGTGCTTAAACAGAGCGAG-3′  
  CTX-M-2R 5′-CCATGAATAAGCAGCTGATTGCCC-3′  
blaCTX-M (CTX-M-8 cluster) CTX-M-8F 5′-ACGCTCAACACCGCGATC-3′  
  CTX-M-8R 5′-CGTGGGTTCTCGGGGATAA-3′  
blaCTX-M (CTX-M-9 cluster) CTX-M-9F 5′-GATTGACCGTATTGGGAGTTT-3′  
  CTX-M-9R 5′-CGGCTGGGTAAAATAGGTCA-3′  
blaTEM TEM-F 5′-ATGAGTATTCAACATTTCCGT-3′ [18]
  TEM-R 5′-TTACCAATGCTTAATCAGTGA-3′  
blaSHV SHV-F 5′-CCGGGTTATTCTTATTTGTCGCT-3′  
  SHV-R 5′-TAGCGTTGCCAGTGCTCG-3′  
blaVEB VEB-F 5′-ACCAGATAGGAGTACAGACATATGA-3′  
  VEB-R 5′-TTCATCACCGCGATAAAGCAC-3′  
blaGES/IBC GES/IBC-F 5′-GTTAGACGGGCGTACAAAGATAAT-3′  
  GES/IBC-R 5′-TGTCCGTGCTCAGGATGAGT-3′  
blaTLA TLA-F 5′-CGCGAAAATTCTGAAATGAC-3′  
  TLA-R 5′-AGGAAATTGTACCGAGACCCT-3′  
blaDHA-1-like DHA-F 5′-GGGGAGATAACGTCTGACCA-3′  
  DHA-R 5′-TAGCCAGATCCAGCAATGTG-3′  
blaCMY-1-like CMY-1F 5′-TCACATCGGCTTCACAGAGC-3′  
  CMY-1R 5′-CCATGGTGATGCTGTCAAAGA-3′  
blaCMY-2-like CMY-2F 5/-CAACACGGTGCAAATCAAAC-3, [19]
  CMY-2R 5′-CATGGGATTTTCCTTGCTGT-3′  
blaACT-1-like ACT-1F 5′-CGTCATGGTCTCGTCCGTTAG-3′  
  ACT-1R 5′-CCTTGACCTCATCCGGTACCT-3′  
qnrA1 to qnrA6 QnrAm-F 5′-AGAGGATTTCTCACGCCAGG-3′  
  QnrAm-R 5′-TGCCAGGCACAGATCTTGAC-3′  
qnrB1 to qnrB6 QnrBm-F 5′-GGMATHGAAATTCGCCACTG-3′  
  QnrBm-R 5′-TTTGCYGYYCGCCAGTCGAA-3′ [20]
qnrS1 to qnrS2 QnrSm-F 5′-GCAAGTTCATTGAACAGGGT-3′  
  QnrSm-R 5′-TCTAAACCGTCGAGTTCGGCG-3′  
aac(6′)-lb AAC(6′)-IbF 5′-TGACCAACAGCAACGATTCC-3′ [21]
  AAC(6′)-IbR 5′-TTAGGCATCACTGCGTGTTC-3′  
armA armA-F 5′-TATGGGGGTCTTACTATTCTGCCTAT-3′  
  armA-R 5′-TCTTCCATTCCCTTCTCCTTT-3′  
rmtA rmtA-F 5′-CTAGCGTCCATCCTTTCCTC-3′  
  rmtA-R 5′-TTTGCTTCCATGCCCTTGCC-3′  
rmtB rmtB-F 5′-TCAACGATGCCCTCACCTC-3′  
  rmtB-R 5′-GCAGGGCAAAGGTAAAATCC-3′ [22]
rmtC rmtC-F 5′-GCCAAAGTACTCACAAGTGG-3′  
  rmtC-R 5′-CTCAGATCTGACCCAACAAG-3′  
rmtD rmtD-F 5′-CTGTTTGAAGCCAGCGGAACGC-3′  
  rmtD-R 5′-GCGCCTCCATCCATTCGGAATAG-3′  
npmA npmA-F 5′-CTCAAAGGAACAAAGACGG-3′  
  npmA-R 5′-GAAACATGGCCAGAAACTC-3′  
Table 2.
Characteristics of urinary E. coli isolates containing genes encoding ESBLs
Strain MICs (mg/L) Susceptibility∗ Trans-Conjugability Antimicrobial resistance genes encoding Phylogenetic group
CAZ CAZ/CA CTX CTX/CA FOX CIP G NN AN ESBL AmpC Qnr AAC(6′)-Ib-cr Methylase
KU05/10441 128 128 >256 >256 >256 >256 R R R + CTX-M-3 CMY-10     armA B2
KU05/17759 64 2 256 1 128 >256 R R R + CTX-M-3 DHA-1 QnrB4 + armA A
KU05/18969 16 1 128 0.3 16 >256 R R I + CTX-M-3     + armA A
KU05/23567 64 8 >256 4 32 >256 R R R + CTX-M-3         B2
KU05/24485 128 256 >256 >256 >256 >256 R R I + CTX-M-3 CMY-10   +   B2
KU05/27254 16 1 >256 0.5 16 >256 R R S + CTX-M-3         D
KU05/14811 32 0.3 256 0.3 32 >256 R R S - CTX-M-15     +   B2
KU05/16013 64 0.5 >256 0.3 8 >256 R R S - CTX-M-15   QnrS1 +   B2
KU05/17301 256 0.5 >256 1 8 >256 R R S - CTX-M-15         B2
KU05/19020 >256 2 >256 1 32 >256 R R S - CTX-M-15     +   B2
KU05/21856 32 0.5 128 0.1 4 16 R R S + CTX-M-15     +   B1
KU05/22571 256 2 >256 1 8 >256 R R S + CTX-M-15     +   A
KU05/23521 64 0.5 128 0.1 8 >256 R R S - CTX-M-15     +   A
KU05/23984 256 2 >256 1 32 >256 R R S + CTX-M-15     +   B2
KU05/24852 128 1 >256 1 32 >256 R R S - CTX-M-15     +   B2
KU05/27014 64 1 256 0.3 4 >256 R R S - CTX-M-15     +   B2
KU05/29305 64 1 >256 0.3 64 >256 R R S - CTX-M-15     +   B2
KU05/29630 64 4 >256 2 256 >256 R R R + CTX-M-15     + rmtB D
KU05/28700 >256 2 >256 0.5 32 64 R R S + CTX-M-15     +   B2
KU05/19028 4 0.5 >256 0.3 32 >256 R S S + CTX-M-14         D
KU05/27080 16 2 >256 1 32 >256 S S S - CTX-M-14         D
KU05/29253 16 2 256 1 16 >256 S R R - CTX-M-14     +   D
KU05/14517 256 1 >256 1 32 >256 R R S - TEM-52     +   B2
KU05/30403 64 1 >256 0.5 16 >256 R R S - CTX-M-14+ CTX-M-15         D
KU05/31131 16 1 >256 0.5 16 >256 R R S + CTX-M-3+ CTX-M-14         D
KU05/15422 256 1 >256 0.5 16 >256 R R I - CTX-M-14+ SHV-12     +   B2
KU05/21306 256 1 128 0.1 4 >256 R R R + CTX-M-3+     + armA B1
                      CTX-M-9+SHV-12          

∗ Antimicrobial susceptibilities of E. coli isolates determined by disk diffusion assay.

Abbreviations: MIC, minimum inhibitory concentration; CAZ, ceftazidime; CA, clavulanic acid; CTX, cefotaxime; FOX, cefoxitin; CIP, ciprofloxacin; G, gentamicin; NN, tobramycin; AN, amikacin; AmpC, AmpC β-lactamase; R, resistant; I, intermediately resistant; S, susceptible; ESBL, extended-spectrum β-lactamase.

Table 3.
Phylogenetic background of the 27 ESBL-producing urinary E. coli isolates
ESBL gene Phylogenetic group Total
A B1 B2 D
blaCTX-M-3 2   3 1 6
blaCTX-M-15 2 1 9 1 13
blaCTX-M-14       3 3
blaTEM-52     1   1
blaCTX-M-14+ blaCTX-M-15       1 1
blaCTX-M-3+ blaCTX-M-14       1 1
blaCTX-M-14+ blaSHV-12     1   1
blaCTX-M-3+blaCTX-M-9+blaSHV-12   1      
Total 4 2 14 7 27

Abbreviation: ESBL, extended-spectrum β-lactamase.

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