Journal List > Korean J Clin Microbiol > v.14(2) > 1038243

TOOLS
Jeong, Son, Shin, Ryu, Hong, Han, and Shin: Characterization of Acinetobacter baumannii Co-producing Carbapenemases OXA-23 and OXA-66, and armA 16S Ribosomal RNA Methylase at a University Hospital in South Korea
Original Article

Korean J Clin Microbiol 2011;14(2):67-73.

Published online: 21 June 2011

DOI: https://doi.org/10.5145/KJCM.2011.14.2.67

Characterization of Acinetobacter baumannii Co-producing Carbapenemases OXA-23 and OXA-66, and armA 16S Ribosomal RNA Methylase at a University Hospital in South Korea

Hye Won Jeong1, Bo Ra Son2, Dong Ick Shin3, Donghee Ryu4, Seung Bok Hong6, Kyudong Han7, Kyeong Seob Shin2, 5

1Department of Internal Medicine, College of Medicine, Chungbuk National University, Cheongju, Korea

2Department of Laboratory Medicine, College of Medicine, Chungbuk National University, Cheongju, Korea

3Department of Neurology, College of Medicine, Chungbuk National University, Cheongju, Korea

4Department of Surgery, College of Medicine, Chungbuk National University, Cheongju, Korea

5BK 21 Chungbuk Biomedical Science Center, College of Medicine, Chungbuk National University, Cheongju, Korea

6Department of Clinical Laboratory Science, Juseong University, Cheongwon, Korea

7Department of Microbiology and Institute of Basic Science, Dankook University, Cheonan, Korea

Correspondence: Kyeong Seob Shin, Department of Laboratory Medicine, College of Medicine, Chungbuk National University, San 62 Gaeshin-dong, Heungduk-gu, Cheongju 361-711, Korea. (Tel) 82-43-269-6240, (Fax) 82-43-271-5243, (E-mail) ksshin@chungbuk.ac.kr

Received 20 August 2010       Revised 18 November 2010       Accepted 30 November 2010

Copyright © 2011 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

In the present study, the resistance mechanisms against carbapenems and aminoglycosides for 23 strains of multidrug-resistant Acinetobacter baumannii isolated at a university hospital were investigated.

Methods

The minimal inhibitory concentrations (MICs) were determined via broth microdilution or Etest. The genes encoding OXA-type carbapenemases and 16S rRNA methylase were identified using multiplex PCR, and the amplified products were sequenced. Conjugation experiments were conducted, and an epidemiologic study was performed using enterobacterial repetitive intergenic consensus (ERIC)-PCR.

Results

In the isolates, the MICs of the tested aminoglycosides, including arbekacin, were ＞1024 μg/ mL; the MICs of aztreonam, cefepime, ceftazidime, and ciprofloxacin ranged from 64 to 128 μg/mL; and the MICs of carbapenem ranged from 32 to 64 μg/ mL, as determined through the broth microdilution test. According to the E-test, the MICs of ampicillin/sulbactam and colistin were 8 and 0.25 to 0.38 μg/ mL, respectively. Sequence analysis confirmed that all of the isolates expressed carbapenemases OXA-23 and OXA-66, as well as armA 16S rRNA methylase. In addition, ISAba1 was identified upstream of the gene encoding OXA-23. OXA-23 and armA were not transferred to Escherichia coli J53 cells in the transconjugation experiments. ERIC-PCR molecular fingerprinting produced a single pattern in all cases.

Conclusion

The co-production of OXA-23 and armA 16S rRNA methylase may be attributed to the multidrug resistance of the A. baumannii isolates in the present study. Stricter surveillance and more rapid detection are necessary to prevent the spread of this type of resistance in the future.

Keywords: Acinetobacter baumannii armA, Outbreak, OXA-23

REFERENCES

1. Villegas MV and Hartstein AI. Acinetobacter outbreaks, 1977∼ 2000. Infect Control Hosp Epidemiol. 2003; 24:284–95.
2. Wilks M, Wilson A, Warwick S, Price E, Kennedy D, Ely A, et al. Control of an outbreak of multidrug-resistant Acinetobacter baumannii-calcoaceticus colonization and infection in an intensive care unit (ICU) without closing the ICU or placing patients in isolation. Infect Control Hosp Epidemiol. 2006; 27:654–8.
3. Walther-Rasmussen J and H⊘iby N. OXA-type carbapenemases. J Antimicrob Chemother. 2006; 57:373–83.
4. Lee K, Kim MN, Choi TY, Cho SE, Lee S, Whang DH, et al. KONSAR Group. Wide dissemination of OXA-type carbapenemases in clinical Acinetobacter spp. isolates from South Korea. Int J Antimicrob Agents. 2009; 33:520–4.
crossref
5. Lee JH, Choi CH, Kang HY, Lee JY, Kim J, Lee YC, et al. Differences in phenotypic and genotypic traits against antimicrobial agents between Acinetobacter baumannii and Acinetobacter genomic species 13TU. J Antimicrob Chemother. 2007; 59:633–9.
crossref
6. Zong Z, Lü X, Valenzuela JK, Partridge SR, Iredell J. An outbreak of carbapenem-resistant Acinetobacter baumannii producing OXA-23 carbapenemase in western China. Int J Antimicrob Agents. 2008; 31:50–4.
crossref
7. Jeon BC, Jeong SH, Bae IK, Kwon SB, Lee K, Young D, et al. Investigation of a nosocomial outbreak of imipenem-resistant Acinetobacter baumannii producing the OXA-23 beta-lactamase in Korea. J Clin Microbiol. 2005; 43:2241–5.
8. Galimand M, Courvalin P, Lambert T. Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother. 2003; 47:2565–71.
9. Lee H, Yong D, Yum JH, Roh KH, Lee K, Yamane K, et al. Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea. Diagn Microbiol Infect Dis. 2006; 56:305–12.
crossref
10. Doi Y, Yokoyama K, Yamane K, Wachino J, Shibata N, Yagi T, et al. Plasmid-mediated 16S rRNA methylase in Serratia marcescens conferring high-level resistance to aminoglycosides. Antimicrob Agents Chemother. 2004; 48:491–6.
11. Bogaerts P, Galimand M, Bauraing C, Deplano A, Vanhoof R, De Mendonca R, et al. Emergence of ArmA and RmtB aminoglycoside resistance 16S rRNA methylases in Belgium. J Antimicrob Chemother. 2007; 59:459–64.
crossref
12. Doi Y, Adams JM, Yamane K, Paterson DL. Identification of 16S rRNA methylase-producing Acinetobacter baumannii clinical strains in North America. Antimicrob Agents Chemother. 2007; 51:4209–10.
13. Doi Y, de Oliveira Garcia D, Adams J, Paterson DL. Coproduction of novel 16S rRNA methylase RmtD and metallo-β-lactamase SPM-1 in a panresistant Pseudomonas aeruginosa isolate from Brazil. Antimicrob Agents Chemother. 2007; 51:852–6.
14. Yan JJ, Wu JJ, Ko WC, Tsai SH, Chuang CL, Wu HM, Lu YJ, Li JD. Plasmid-mediated 16S rRNA methylases conferring high-level aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae isolates from two Taiwanese hospitals. J Antimicrob Chemother. 2004; 54:1007–12.
crossref
15. Marques MB, Brookings ES, Moser SA, Sonke PB, Waites KB. Comparative in vitro antimicrobial susceptibilities of nosocomial isolates of Acinetobacter baumannii and synergistic activities of nine antimicrobial combinations. Antimicrob Agents Chemother. 1997; 41:881–5.
crossref
16. La Scola B, Gundi VA, Khamis A, Raoult D. Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species. J Clin Microbiol. 2006; 44:827–32.
18. Lee K, Chong Y, Shin HB, Kim YA, Yong D, Yum JH. Modified Hodge and EDTA-disk synergy tests to screen metallo-β-lactamase-producing strains of Pseudomonas and Acinetobacter species. Clin Microbiol Infect. 2001; 7:88–91.
19. Shin KS, Son BR, Hong SB, Kim J. Dipicolinic acid-based disk methods for detection of metallo-β-lactamase-producing Pseudomonas spp. and Acinetobacter spp. Diagn Microbiol Infect Dis. 2008; 62:102–5.
crossref
20. Yum JH, Yong D, Lee K, Kim HS, Chong Y. A new integron carrying VIM-2 metallo-β-lactamase gene cassette in a Serratia marcescens isolate. Diagn Microbiol Infect Dis. 2002; 42:217–9.
crossref
21. Poirel L, Naas T, Nicolas D, Collet L, Bellais S, Cavallo JD, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother. 2000; 44:891–7.
22. Lee K, Yum JH, Yong D, Lee HM, Kim HD, Docquier JD, et al. Novel acquired metallo-β-lactamase gene, bla(SIM-1), in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob Agents Chemother. 2005; 49:4485–91.
23. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents. 2006; 27:351–3.
crossref
24. Segal H, Garny S, Elisha BG. Is IS(ABA-1) customized for Acinetobacter ? FEMS Microbiol Lett. 2005; 243:425–9.
25. Jacoby GA and Han P. Detection of extended-spectrum β-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli. J Clin Microbiol. 1996; 34:908–11.
26. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res. 1991; 19:6823–31.
27. Donald HM, Scaife W, Amyes SG, Young HK. Sequence analysis of ARI-1, a novel OXA β-lactamase, responsible for imipenem resistance in Acinetobacter baumannii 6B92. Antimicrob Agents Chemother. 2000; 44:196–9.
28. Chu YW, Cheung TK, Chu MY, Lo JY, Dijkshoorn L. OXA-23-type imipenem resistance in Acinetobacter baumannii in Hong Kong. Int J Antimicrob Agents. 2009; 34:285–6.
crossref
29. Cho YJ, Moon DC, Jin JS, Choi CH, Lee YC, Lee JC. Genetic basis of resistance to aminoglycosides in Acinetobacter spp. and spread of armA in Acinetobacter baumannii sequence group 1 in Korean hospitals. Diagn Microbiol Infect Dis. 2009; 64:185–90.
crossref
30. Zhou H, Du XX, Yang Q, Zhou JY, Yu YS, Li LJ. Study on carbapenemase and 16S rRNA methylase of imipenem-resistant Acinetobacter baumannii. Zhonghua Liu Xing Bing Xue Za Zhi. 2009; 30:269–72.
31. Zhou H, Yang Q, Yu YS, Wei ZQ, Li LJ. Clonal spread of imipenem-resistant Acinetobacter baumannii among different cities of China. J Clin Microbiol. 2007; 45:4054–7.
32. Kim JW, Heo ST, Jin JS, Choi CH, Lee YC, Jeong YG, et al. Characterization of Acinetobacter baumannii carrying bla(OXA-23), bla(PER-1) and armA in a Korean hospital. Clin Microbiol Infect. 2008; 14:716–8.

Fig. 1.
The finding of DNA fingerprints by ERIC-PCR of Acinetobacter baumannii clinical isolates. Clinical isolates with outbreak (lanes 2∼9) exhibits single DNA fingerprinting pattern but non-outbreak strains (lanes 10∼13) showed different patterns. Lane 1, molecular size marker (100 bp ladder); lanes 2∼9, clinical isolates of A. baumannii with outbreak; lanes 10∼13, clinical isolates of A. baumannii with non-outbreak strain, including cabapenem resistant A. baumannii isolated from other two University hospital; lane 14, molecular size marker (100 bp ladder).
kjcm-14-67f1.tif
Table 1.
Primers used in this study
Primer Sequence 5′→3′ Target Reference
Ac1055 F GTG ATA ARA TGG CBG GTC GT rpoB [16]
Ac1598 R CGB GCR TGC ATY TTG TCR T    
OXA-23-like F GAT CGG ATT GGA GAA CCA GA blaOXA-23-like [23]
OXA-23-like R ATT TCT GAC CGC ATT TCC AT    
OXA-24-like F GGT TAG TTG GCC CCC TTA AA blaOXA-24-like [23]
OXA-24-like R AGT TGA GCG AAA AGG GGA TT    
OXA-51-like F TAA TGC TTT GAT CGG CCT TG blaOXA-51-like [23]
OXA-51-like R TGG ATT GCA CTT CAT CTT GG    
OXA-58-like F AAG TAT TGG GGC TTG TGC TG blaOXA-58-like [23]
OXA-58-like R CCC CTC TGC GCT CTA CAT AC    
ISAba1F CAC GAA TGC AGA AGT TG ISAba1/blaOXA-23-like [24]
OXA-23-like R ATT TCT GAC CGC ATT TCC AT    
OXA-51-like R TGG ATT GCA CTT CAT CTT GG ISAba1/blaOXA-51-like [24]
IMP-1 F CAT GGT TTG GTG GTT CTT GT blaIMP-1 [20]
IMP-1 R ATA ATT TGG CGG ACT TTG GC    
VIM-2 F ATG TTC AAA CTT TTG AGT AAG blaVIM-2 [21]
VIM-2 R CTA CTC AAC GAC TGA GCG    
SIM-1 F TAC AAG GGA TTC GGC ATCG blaSIM-1 [22]
SIM-2 R TAA TGG CCT GTT CCC ATG TG    
ArmA F CAA ATG GAT AAG AAT GAT GTT armA [8]
ArmA R TTA TTT CTG AAA TCC ACT    
RtmB F ATG AAC ATC AAC GAT GCC CT rtmB [14]
RtmB R CCT TCT GAT TGG CTT ATC CA    
ERIC1 R ATGTAAGCTCCTGGGGATTCAC ERIC sequences [26]
ERIC2 AAGTAAGTGACTGGGGTGAGCG    

Abbreviation: ERIC, enterobacterial repetitive intergenic consensus.

Table 2.
Minimal inhibitory concentration (MIC) of Acinetobacter baumannii clinical isolates
No. Minimal inhibitory concentration (μg/mL)
CTZ FEP ATM A/S∗ P/T∗ IMP MEM CIP CS∗
88 128 64 128 8 ＞256 32 64 64 0.25
89 64 64 128 8 ＞256 32 64 64 0.25
91 128 64 128 8 ＞256 32 64 64 0.25
92 128 64 128 8 ＞256 32 64 64 0.38
94 128 64 128 8 ＞256 32 32 64 0.25
95 64 64 128 8 ＞256 32 64 64 0.25
96 64 64 128 8 ＞256 32 32 64 0.25
98 64 64 128 8 ＞256 32 64 64 0.25
103 128 64 128 8 ＞256 32 64 64 0.25
104 128 64 128 8 ＞256 32 64 64 0.25
105 128 64 128 8 ＞256 64 64 64 0.25
106 64 64 128 8 ＞256 32 64 64 0.25
201 128 64 128 8 ＞256 32 64 64 0.25
202 128 64 128 8 ＞256 64 64 64 0.25
203 64 64 128 8 ＞256 32 32 64 0.25
205 64 64 128 8 ＞256 32 64 64 0.25
207 128 64 128 8 ＞256 32 64 64 0.38
208 128 64 128 8 ＞256 32 64 64 0.25
209 128 128 128 8 ＞256 32 32 64 0.25
210 128 128 128 8 ＞256 32 64 64 0.25
211 128 128 128 8 ＞256 32 64 64 0.25
212 64 64 128 8 ＞256 32 64 64 0.25
213 128 64 128 8 ＞256 32 32 64 0.25

MIC of four aminoglycoside antimicrobial agents including amikacin, gentamicin, tobramycin and arbekacin was ＞1,024μg/mL in all of the isolates.

∗ MIC was determined by Etest (AB BIODISK, Solna, Sweden). Abbreviations: CTZ, ceftazidime; FEP, cefepime; ATM, aztreonam; CIP, ciprofloxacin; A/S, ampicillin/sulbactam; P/T, piperacillin/tazobactam; IMP, imepenem; MEM, meropenem; CS, colistin.

Table 3.
Clinical information and genetic characteristics of Acinetobacter baumannii clinical isolates
No Isolation date Isolation ward Specimen OXA-like∗ ISAba1/ OXA-23† armA‡ ERIC-PCR
23 51
88 07-05-07 ICU Sputum ＋ ＋ ＋ ＋ A
89 07-05-08 ICU Sputum ＋ ＋ ＋ ＋ A
91 07-05-11 ICU Ascitic F ＋ ＋ ＋ ＋ A
92 94 07-05-15 07-05-28 ICU ICU Sputum Ascitic F ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ AA
95 07-05-25 ICU Sputum ＋ ＋ ＋ ＋ A
96 07-05-30 ICU Sputum ＋ ＋ ＋ ＋ A
98 07-06-20 ICU Sputum ＋ ＋ ＋ ＋ A
103 07-07-05 A-W Sputum ＋ ＋ ＋ ＋ A
104 07-07-09 B-W Ascitic F ＋ ＋ ＋ ＋ A
105 07-07-10 C-W BA ＋ ＋ ＋ ＋ A
106 07-07-10 C-W BA ＋ ＋ ＋ ＋ A
201 07-07-19 ICU BA ＋ ＋ ＋ ＋ A
202 07-07-26 D-W BA ＋ ＋ ＋ ＋ A
203 07-07-24 E-W Urine ＋ ＋ ＋ ＋ A
205 07-07-24 B-W BA ＋ ＋ ＋ ＋ A
207 07-09-27 ICU Sputum ＋ ＋ ＋ ＋ A
208 07-09-27 ICU Blood ＋ ＋ ＋ ＋ A
209 07-10-03 ICU Sputum ＋ ＋ ＋ ＋ A
210 07-10-11 ICU Sputum ＋ ＋ ＋ ＋ A
211 212 07-10-11 07-10-13 ICU E-W Blood Other ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋ AA
213 07-10-29 ICU Sputum ＋ ＋ ＋ ＋ A

∗ blaOXA-23 like and blaOXA-51 like was positive by multiplex PCR, and the amplified products were respectively confirmed to OXA-23 and OXA-66 type by sequencing analysis in eight isolates selected arbitrarily.

† ISAb1 was located to upstream of blaOXA-23 (separated to 27 bp) but was not presented at upstream of blaOXA-66.

‡ The amplified products were confirmed to be armA gene by the sequencing analysis. Abbreviations: ICU, intensive care unit; W, ward; F, fluid; BA, bronchial aspirate.

TOOLS
Similar articles