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Kim, Sung, Park, Kwon, and Koo: Coproduction of qnrB and armA from Extended-Spectrum β-lactamase-producing Klebsiella pneumoniae
Original Article

Korean J Leg Med 2007;27(6):428-436.

Published online: 14 February 2007

DOI: https://doi.org/10.3343/kjlm.2007.27.6.428

Coproduction of qnrB and armA from Extended-Spectrum β-lactamase-producing Klebsiella pneumoniae

Moon Hee Kim, M.D.1, Ji Youn Sung2, Jong Woo Park, M.D.2, Gye Cheol Kwon, M.D.2, Sun Hoe Koo, M.D.2

1Department of Laboratory Medicine, Sun General Hospital, Daejeon, Korea

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

Received 2 August 200724 October 2007       Accepted 25 October 2007

Copyright © 2007 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

Multidrug-resistant Enterobacteriaceae is a worldwide problem. Although various resistance mechanisms have been recognized with increasing frequency, only a few cases of triple resistance of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported. This study was designed to evaluate the coexistence of qnr (qnrA, qnrB, and qnrS) and 16S rRNA methylase (armA, rmtA, rmtB, and rmtC) in ESBL-producing K. pneumoniae.

Methods

We tested 44 isolates of ESBL-producing K. pneumoniae at Chungnam National University Hospital from March to September 2006. Antimicrobial susceptibilities were tested by broth microdilution method, and transconjugation test was performed using E. coli J53 with azide resistance. Search for qnr (qnrA, qnrB, and qnrS) and 16S rRNA methylase (armA, rmtA, rmtB, and rmtC) genes was conducted by PCR amplification, and the genotypes were determined by direct nucleotide sequence analysis of the amplified products. Epidemiologic study was performed by Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR).

Results

All ESBL-positive strains produced qnrB; however, armA was detected in 68.2%. The coproduction rate of qnrB and armA in ESBL-producing K. pneumoniae was 68.2%. Two types (A and B) were dominant in ERIC-PCR results.

Conclusions

K. pneumoniae producing qnrB, armA, and ESBL are spreading widely.

Keywords: Extended-spectrum β-lactamase, qnrB, armA, Klebsiella pneumoniae

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Fig. 1.
Enterobacterial repetitive intergenic consensus-PCR patterns of genomic DNA from clinical isolates of K. pneumoniae harboring ESBLs. Lane M is 1kb DNA size marker. Among the five different banding patterns (A, B, C, D, E), A and B patterns are dominant. Thirty nine of 44 extended-spectrum β-lactamase -producing clinical isolates show A or B patterns.
kjlm-27-428f1.tif
Table 1.
Oligonucleotides used as primers for amplification and sequencing in this study
Enzymes Primer pairs Target Sequence (5′-3′) Amplicon size (bp) Reference
β-lactamase TEM F∗ blaTEM and variants ATGAGTATTCAACATTTCCGT 861 28
  TEM R†   TTACCAATGCTTAATCAGTGA    
  SHV F blaSHV and variants CCGGGTTATTCTTATTTGTCGCT 831 28
  SHV R   TAGCGTTGCCAGTGCTCG    
  CTX-M1F blaCTX-M-1 cluster AGTTCACGCTGATGGCGACG 676 23
  CTX-M1R   AACCCAGGAAGCAGGCAGTCC    
  CTX-M9F blaCTX-M-9 cluster GATTGACCGTATTGGGAGTTT 947 28
  CTX-M9R   CGGCTGGGTAAAATAGGTCA    
Qnr qnrA F qnrA GGGTATGGATATTATTGATAAAG 660 29
  qnrA R   CTAATCCGGCAGCACTATTA    
  qnrB F qnrB1-qnrB6 variants GGMATHGAAATTCGCCACTG 264 29
  qnrB R   TTTGCYGYYCGCCAGTCGAA    
  qnrS F qnrS AGTGATCTCACCTTCACCGC 550 29
  qnrS R   CAGGCTGCAATTTTGATACC    
16S rRNA methylase armA F armA AGGTTGTTTCCATTTCTGAG 776 23
  armA R   TCTCTTCCATTCCCTTCTCC    
  rmtA F rmtA CTAGCGTCCATCCTTTCCTC 635 23
  rmtA R   TTTGCTTCCATGCCCTTGCC    
  rmtB F rmtB CCCAAACAGACCGTAGAGGC 769 23
  rmtB R   CTCAAACTCGGCGGGCAAGC    
  rmtC F rmtC CGAAGAAGTAACAGCCAAAG 700 30

∗ F, forward;

† R, reverse.

Table 2.
Characteristics of ESBL-producing K. pneumoniae harboring qnrB, qnrS, and armA genes
Isolates MIC of agents (μg/mL) β-lactamase qnr 16S rRNA methylase ERIC type
AMK GEN CAZ FEP CTX CIP NOR
K-1 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-2 8 ≥16 ≥64 2 8 2 2 SHV-12 qnrB4 armA B
K-3 ≥64 ≥16 ≥64 ≤1 8 ≥4 8 SHV-12 qnrB4 armA B
K-4 4 1 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-10 ≥64 ≥16 ≥64 ≤1 ≥64 0.5 2 SHV-12 qnrB4 armA C
K-12 16 2 ≥64 ≤1 4 ≤0.25 ≤0.5 SHV-12 qnrB4 armA B
K-16 ≥64 ≥16 ≥64 2 4 ≥4 ≥16 SHV-12 qnrB4 armA A
K-17 ≥64 8 ≥64 ≤1 ≥64 ≥4 ≥16 SHV-12 qnrB4   B
K-19 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA B
K-20 ≥64 8 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12, CTX-M3 qnrB4 armA B
K-24 ≥64 ≥16 ≥64 2 16 ≥4 ≥16 SHV-12 qnrB4 armA C
K-25 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4   A
K-27 ≤2 4 ≤1 ≤1 8 1 2 CTX-M9 qnrB4   D
K-32 ≥64 ≥16 ≥64 ≤1 32 ≥4 ≥16 CTX-M9 qnrB4   B
K-38 ≥64 ≥16 ≥64 ≤1 32 ≥4 ≥16 SHV-12 qnrB4   B
K-42 ≥64 8 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12, CTX-M9 qnrB4   B
K-43 16 2 ≥64 2 8 ≥4 ≥16 SHV-12 qnrB4   B
K-45 16 ≥16 ≥64 ≤1 32 ≤0.25 2 SHV-12 qnrB4   C
K-46 4 ≤1 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-85 ≥64 ≥16 ≥64 4 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-86 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-88 8 ≥16 ≥64 ≤1 8 1 2 SHV-12 qnrB4   B
K-89 ≥64 ≥16 ≥64 1 32 ≥4 ≥16 SHV-12 qnrB4   B
K-91 ≥64 ≥16 ≥64 4 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-93 ≥64 ≥16 ≥64 4 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-95 ≥64 ≥16 ≥64 16 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-96 ≥64 ≥16 ≥64 ≤1 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-99 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-100 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-102 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-146 ≥64 ≥16 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12 qnrB4   E
K-148 ≥64 ≥16 ≥64 ≤1 2 ≥4 ≥16 SHV-12 qnrB4 armA B
K-150 ≤2 ≥16 ≤1 ≤1 ≥64 2 8 SHV-12, CTX-M9 qnrS, qnrB4   B
K-152 ≥64 ≥16 ≥64 ≤1 4 ≥4 ≥16 SHV-12 qnrB4 armA A
K-153 ≥64 8 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-154 ≥64 ≥16 ≥64 ≥64 ≥64 ≥4 ≥16 SHV-12, CTX-M3 qnrB4 armA B
K-155 ≥64 ≥16 ≥64 2 16 ≥4 ≥16 SHV-12 qnrB4 armA A
K-156 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-159 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-160 ≥64 ≥16 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA A
K-161 ≤2 ≥16 2 2 ≥64 0.5 2 SHV-12, CTX-M9 qnrS, qnrB4   B
K-164 ≤2 8 ≥64 2 16 ≥4 8 SHV-12 qnrB4   B
K-166 ≥64 ≥16 ≥64 8 ≥64 ≥4 ≥16 SHV-12 qnrB4 armA A
K-167 4 2 ≥64 ≤1 8 ≥4 ≥16 SHV-12 qnrB4 armA B

Abbreviations: MIC, minimum inhibitory concentration, AMK, amikacin; GEN, gentamicin; CAZ, ceftazidime; FEP, cefepime; CTX, cefotaxime; CIP, ciprofloxacin; NOR, norfloxacin; ERIC, Enterobacterial repetitive intergenic consensus.

Table 3.
Activities of antimicrobial agents against ESBL producing K. pneumoniae harboring qnrB, qnrS, and armA genes
Antibiotic resistant genes (N of isolate) AMK GEN CAZ CTX CIP NOR
MIC range MIC50 MIC90 MIC range MIC50 MIC90 MIC range MIC50 MIC90 MIC range MIC50 MIC90 MIC range MIC50 MIC90 MIC range MIC50 MIC90
blaSHV-12, qnrB4 (9) ≤0.25->64 >64 >64 2->16 >16 >16 ≥64 >64 >64 8->64 32 >64 ≤0.01->4 >4 >4 ≤0.01->16 >16 >16
blaSHV-12, qnrB4, armA (28) 4->64 >64 >64 ≤0.1->16 >16 >16 ≥64 >64 >64 2->64 8 >64 ≤0.01->4 >4 >4 ≤0.01->16 >16 >16
blaSHV-12, blaCTX-M3, qnrB4, armA (2) >64 >64 >64 8->16 8 >16 ≥64 >64 >64 ≥64 >64 >64 >4 >4 >4 >16 >16 >16
blaSHV-12, blaCTX-M9, qnrB4 (1) >64 >64 >64 8 8 8 ≥64 >64 >64 ≥64 >64 >64 >4 >4 >4 >16 >16 >16
blaSHV-12, blaCTX-M9, qnrB4, qnrS (2) ≤0.25 ≤0.25 ≤0.25 >16 >16 >16 ≤0.15-2 1 2 ≥64 >64 >64 0.5-2 1 2 2-8 4 8
blaCTX-M9, qnrB4 (2) ≤0.25->64 32 >64 4->16 8 >16 ≤0.15-≥64 32 >64 8-32 16 >32 1-≥4 2 >4 2->16 8 >16
Total (44) ≤0.25->64 >64 >64 ≤0.1->16 >16 >16 ≤0.15-≥64 >64 >64 ≤0.1-≥64 16 >64 ≤0.01->4 >4 >4 ≤0.01->16 >16 >16

Abbreviations: See Table 2.

Table 4.
Distributions of qnrB, qnrS and armA determinants according to ESBL among clinical isolates of K. pneumoniae
ESBL type (N of isolate) N (%) of isolates with
armA qnrB4 qnrS
SHV-12 (37) 28 (75.7) 37 (100)  
SHV-12, CTX-M-9 (3)   3 (100) 2 (66.7)
SHV-12, CTX-M-3 (2) 2 (100) 2 (100)  
CTX-M-9 (2)   2 (100)  
Total (44) 30 (68.2) 44 (100) 2 (4.5)

Abbreviation: ESBL, extended-spectrum β-lactamase.

Table 5.
Resistance profile of donor strains and transconjugants
Isolates MIC of agents (μg/mL) β-lactamase qnr 16S rRNA methylase
AMK GEN CAZ CTX CIP
Donors (state of origin)                
 K-3 ≥64 ≥16 ≥64 8 ≥4 SHV qnrB armA
 K-4 4 1 ≥64 8 ≥4 SHV qnrB armA
 K-12 16 2 ≥64 4 0.25 SHV qnrB armA
 K-17 ≥64 8 ≥64 ≥64 ≥4 SHV qnrB armA
 K-20 ≥64 8 ≥64 ≥64 ≥4 SHV qnrB armA
 K-24 ≥64 ≥16 ≥64 16 ≥4 SHV qnrB armA
 K-100 ≥64 ≥16 ≥64 8 ≥4 SHV qnrB armA
 K-102 ≥64 ≥16 ≥64 8 ≥4 SHV qnrB armA
 K-159 ≥64 ≥16 ≥64 8 ≥4 SHV qnrB armA
 K-160 ≥64 ≥16 ≥64 4 ≥4 SHV qnrB armA
J53 transconjugants                
 K-3′ ≥64 8 ≥64 4 ≥4 SHV qnrB armA
 K-4′ 2 1 ≥64 1 0.25 SHV qnrB armA
 K-12′ 4 2 16 2 0.01 SHV qnrB  
 K-17′ 4 2 16 1 0.25 SHV qnrB armA
 K-20′ 4 2 16 1 0.25 SHV qnrB armA
 K-24′ 2 2 16 1 2 SHV qnrB armA
 K-100′ 8 0.1 ≥64 16 0.01 SHV qnrB armA
 K-102′ 2 0.1 ≥64 32 0.01 SHV qnrB armA
 K-159′ 1 0.1 16 1 0.1 SHV qnrB armA
 K-160′ 2 2 16 1 10 SHV qnrB armA
E. coli J53 0.25 0.1 0.15 0.06 0.01      

Abbreviations: See Table 2.

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