kjcm Korean Journal of Clinical Microbiology Korean J Clin Microbiol 1229-0025 Korean Journal of Clinical Microbiology 10.5145/KJCM.2010.13.1.19 kjcm-13-19 Original Article Molecular and Phenotypic Characteristics of 16S rRNA Methylase-producing Gram-negative Bacilli Lee Hyukmin 1 Koh Eun-Mi 2 Kim Chang Ki 3 Yum Jong Hwa 4 Lee Kyungwon 5 Chong Yunsop 5 Department of Laboratory Medicine, Kwandong University College of Medicine, Goyang, Korea Incheon Blood Center, Korean Red Cross, Incheon, Korea Korean Institute of Tuberculosis, Seoul, Korea Department of Clinical Laboratory Science, Dongeui University, Busan, Korea Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea Correspondence: Kyungwon Lee, Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, 134, Shinchon-dong, Seodaemun-gu, Seoul 120-752, Korea. (Tel) 82-2-2228-2446, (Fax) 82-2-313-0908, (E-mail) leekcp@yuhs.ac 32010 2132010 131 1926 06042009 25112009 20122009 Copyright © 2010 Korean Journal of Clinical Microbiology 2010 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 a novel plasmid-mediated resistant mechanism that conferred high-level resistance to aminoglycoside via methylation of 16S rRNA was reported. The aims of this study were to determine the prevalence of the 16S rRNA methylase genes and to characterize the coresistance to other antibiotics in Gram-negative bacilli.

 Methods

Consecutive non-duplicate Gram-negative bacilli were isolated from clinical specimens at a Korean secondary- and tertiary-care hospital from July 2006 to June 2007. The antimicrobial susceptibility was tested by the CLSI agar dilution method, and PCR was performed to detect the 16S rRNA methylase genes in the arbekacin-resistant isolates.

 Results

In Gram-negative bacilli, the proportions of 16S rRNA methylase gene-positive isolates were 5% (75/1,471) in the secondary-carehospital and 4% (48/1,251) in the tertiary-care hospital, and the positive rates by species were 1% Escherichiae coli 16% (10/1,062), Klebsiella pneumoniae 16% (75/ 460), K. oxytoca 2% (1/44), Citrobacter spp. 9% (7/ 82), Enterobacter spp. 2% (4/181), Serratia marcescens 6% (6/100), Proteus miriabilis 4% (2/57), Achromobacter xylosoxidans 20% (1/5), Pseudomonas aer- uginosa ＜1% (1/505), Acinetobacter spp. 10% (11/ 112), and Stenotrophomonas maltophilia 2% (1/66), respectively. Among 16S rRNA methylase-positive isolates from secondary- and tertiary-care hospitals,93% (70/75) and 90% (43/48), respectively, were armA positive, and others, except one rmtA positive isolate, were positive for the rmtB gene, according to PCR results. The rates of ESBL-positive and cefoxitin-resistant K. pneumoniae were 59% and 92%, respectively. In addition, 91% of 16S rRNA methylase-producing K. pneumoniae were positive for qnrB. There were no MBL producers among 16S rRNA methylase-producing Pseudomonas and Acinetobacter species.

 Conclusion

The novel aminoglycoside-resistant mechanisms involving16S rRNA methylase were prevalent and widely distributed among Gram-negative bacilli in Korea, and other resistance mechanisms were commonly associated with 16S rRNA methylase-mediated resistance in Korea.

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Primers used for PCR detection of 16S rRNA methylase genes, qnr, and qepA genes

Name Nucleotide sequence (5’→3’) Product size (bp) GenBank accession No.
armA F armA R AGG TTG TTT CCA TTT CTG AG TCT CTT CCA TTC CCT TCT CC 590 AB116388
rmtA F rmtA R CTA GCG TCC ATC CTT TCC TC TTT GCT TCC ATG CCC TTG CC 635 AB083212
rmtB F rmtB R CCC AAA CAG ACC GTA GAG GC CTC AAA CTC GGC GGG CAA GC 584 AB103506
rmtC F rmtC R CGA AGA AGT AAC AGC CAA AG ATC CCA ACA TCT CTC CCA CT 711 AB194779
rmtD F rmtD R ATG AGC GAA CTG AAG GAA AAA CTG C GCT CCA AAA GCG GCA GCA CCT TA 532 DQ914960
qnrA F qnrA R AGA GGA TTT CTC ACG CCA GG TGC CAG GCA CAG ATC TTG AC 580 qnrA1-qnrA6∗
qnrB F qnrB R GGA ATT GAA ATT CGC CAC TG TTT GCC GCC CGC CAG TCG AA 264 qnrB1-qnrB6∗
qnrS F qnrS R GCA AGT TCA TTG AAC AGG GT TCT AAA CCG TCG AGT TCG GCG 428 qnrS1-qnrS2∗
qepA F qepA R CCG ACA GGC CCA CGA CGA GGA TGC TCG GCG GCG TGT TGC TGG AGT TCT 549 AB263754

Primers designed for multiplex PCR of various qnr genes by Cattoir V, et al.12.

The rates and distributions of 16S rRNA methylase in gram-negative bacilli isolated at a secondary- and tertiary-care hospital from 2006 to 2007

Bacterial species Secondary-care hospital (Myongji Hospital) Tertiary-care hospital (Severance Hospital)
No. isolated No. of PCR positive for No. isolated No. (%) of PCR positive for
Total (%) armA rmtB armA＋rmtB Total (%) armA rmtB armA＋rmtB
E. coli 646 8 (1) 8 0 0 416 2 (＜1) 0 1 1
K. pneumoniae 245 51 (21) 49 0 2 215 24 (11) 24 0 0
K. oxytoca     ND     44 1 (2) 1 0 0
Citrobacter spp. 37 3 (8) 3 0 0 45 4 (10) 2 2 0
Enterobacter spp. 129 2 (2) 2 0 0 52 2 (4) 2 0 0
S. marcescens 52 4 (8) 4 0 0 48 2 (4) 2 0 0
P. mirabilis 57 2 (4) 2 0 0     ND    
M. morganii 27 1 (4) 1 0 0     ND    
Providencia spp. 21 3 (14) 0 2 1     ND    
A. xylosoxidans∗     ND     5 1 (20) 0 0 0
P. aeruginosa 257 1 (＜1) 1 0 0 248 0 (0) 0 0 0
Acinetobacter spp.     ND     112 11 (10) 11 0 0
S. maltophilia     ND     66 1 (2) 1 0 0
Total 1,471 75 (5) 70 2 3 1,251 48 (4) 44 3 1

A. xylosoxidans: rmtA positive. Abbreviation: ND, not determined.

Comparisons of MICs (μg/mL) of antibiotics for 16S rRNA methylase-positive and -negative gram-negative bacilli

Antimicrobial agents 16S rRNA methylase-positive 16S rRNA methylase-negative
MIC range MIC50 MIC90 %R MIC range MIC50 MIC90 %R
Enterobateriaceae (133)
 Ceftazidime 0.12∼＞128 64 ＞128 66 1∼＞128 32 ＞128 63
 Cefoxitin 0.5∼＞128 ＞128 ＞128 85 8∼＞128 32 ＞128 68
 Imipenem 0.06∼32 0.25 2 4 ≤0.06∼4 0.25 1 0
 Levofloxacin 0.06∼＞128 64 128 81 0.12∼＞128 2 128 36
 Arbekacin ＞128 ＞128 ＞128 100 2∼＞128 16 ＞128 50
 Amikacin ＞128 ＞128 ＞128 100 8∼＞128 16 ＞128 50
 Gentamicin ＞128 ＞128 ＞128 100 8∼＞128 128 ＞128 100
 Tobramycin ＞128 ＞128 ＞128 100 32∼＞128 64 ＞128 100
Pseudomonas (9) and Acinetobacter (13)
 Ceftazidime 128∼＞128 ＞128 ＞128 100 2∼＞128 16 ＞128 45
 Imipenem 1∼8 4 4 0 1∼32 16 32 72
 Levofloxacin 4∼64 8 32 63 2∼＞128 64 128 90
 Arbekacin ＞128 ＞128 ＞128 100 2∼＞128 64 ＞128 63
 Amikacin ＞128 ＞128 ＞128 100 4∼＞128 64 ＞128 90
 Gentamicin ＞128 ＞128 ＞128 100 2∼＞128 ＞128 ＞128 90
 Tobramycin ＞128 ＞128 ＞128 100 0.5∼＞128 128 ＞128 90

Comparisons of the rates of β-lactam resistance and quinolone resistance in 16S rRNA methylases-positive and -negative gram-negative bacilli

Bacterial species (No. of isolates) No. (%) of other resistance mechanisms
ESBL PABL qnrA qnrB qnrS qepA∗
16S rRNA methylase-positive
 E. coli (10) 7 (70) 7 (70) 0 (0) 2 (20) 1 (10) 1 (10)
 K. pneumoniae (75) Other Enterobacteriaceae (23) 44 (59) 13 (57) 69 (92) NT 1 (2) 2 (9) 68 (91) 9 (39)† 0 0 0 1 (4)
 Pseudomonas spp. and Acinetobacter spp. (12) 0‡ NT 0 1 (8) 0 0
16S rRNA methylase-negative
 E. coli (4) 2 (50) 1 (25) 0 0 0 1 (25)
 K. pneumoniae (14) 6 (43) 9 (64) 0 8 (57) 1 (7) 0
 Other Enterobacteriaceae (4) 1 (25) NT 0 1 (25) 0 0
 Pseudomonas spp. and Acinetobacter spp. (11) NT NT 0 4 (36) 0 0

2 of 3 qepA-positive strains were rmtB-positive strains

7 qnrB-positive strains were Citrobacter species

Metallo-β-lactamase production tested in P. aeruginosa.