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Annals of Clinical & Laboratory Science 40:43-48 (2010)
© 2010 Association of Clinical Scientists

Role of OXA-23 and AdeABC Efflux Pump for Acquiring Carbapenem Resistance in an Acinetobacter baumannii Strain Carrying the blaOXA-66 Gene

Yangsoon Lee1, Jong Hwa Yum2, Chang-Ki Kim3, Dongeun Yong1, Eun Hee Jeon1, Seok Hoon Jeong1, Jee Young Ahn4 and Kyungwon Lee1
1 Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul; 2 Department of Clinical Laboratory Science, Dong-eui University, Busan; 3 Korean Institute of Tuberculosis, Seoul; and 4 Department of Laboratory Medicine, Soonchunhyang University College of Medicine, Gumi, Korea

Address correspondence to Dr. Seok Hoon Jeong, Department of Laboratory Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, 120-752 Seoul, Korea; tel 82-2-2228-2448; fax 82-2-313-0908; e-mail kscpjsh{at}yuhs.ac.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 
This study was performed to determine the mechanisms for acquiring carbapenem resistance in six clinical isolates of Acinetobacter baumannii. All isolates showed similar SmaI-macrorestriction patterns with less than 3 band differences by PFGE. The isolates showed a high level resistance (>32 mg/L) to both imipenem and meropenem by Etest. Phe-Arg-β-naphthylamide lowered the MICs of carbapenems. Real-time PCR experiments showed that expression levels of the adeB gene in the six A. baumannii isolates were 10- to 40-times higher than those of imipenem-susceptible strains. Direct sequencing of PCR products showed that all isolates carried the blaOXA-23 gene, which was preceded by ISAba1. The blaOXA-23 probe hybridized with approximately 500-kb I-CeuI chromosomal fragments, but not with a plasmid. These findings suggest that overexpression of the AdeABC efflux pump as well as chromosome-borne OXA-23 may play a role in acquiring carbapenem resistance in our A. baumannii isolates.

Keywords: carbapenem, meropenem, imipenem, Acinetobacter baumannii, antimicrobial resistance


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 
The rapid global dissemination of Acinetobacter spp. resistant to carbapenems represents a significant clinical threat [1,2]. Carbapenem resistance in Acinetobacter spp. has mainly been ascribed to enzymatic degradation by metallo-β-lactamses (MBLs) or OXA carbapenemases such as OXA-23, OXA-24, OXA-58, and OXA-51 groups [3,4]. The OXA-51 group consists of a large number of closely related variants including OXA-66, -69, and -83 [5,6]. Chromosome-borne blaOXA-51-like genes are ubiquitous in Acinetobacter baumannii. The blaOXA-51-like genes play a role in carbapenem resistance when an ISAba1 precedes the gene, while they have little effect on carbapenem susceptibility in the absence of this insertion sequence [7]. Non-enzymatic mechanisms including overexpression of efflux pumps and changes in outer membrane proteins (OMPs) or penicillin-binding proteins may play a role in acquiring carbapenem resistance [5]. AdeABC, a resistance-nodulation-division (RND) efflux pump, has come to the forefront as a key player in acquiring antimicrobial resistance in A. baumannii. However, its effects on carbapenem resistance are less clear [8]. We studied an outbreak of carbapenem-resistant A. baumannii carrying the blaOXA-66 gene, a member of blaOXA-51-like gene cluster. Our findings suggest that OXA-23 carbapenemase and overexpression of the AdeABC efflux pump may be associated with carbapenem resistance in A. baumannii.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgement
 References
 
Bacterial strains.  Between January and May 2007, A. baumannii isolates exhibiting resistance to carbapenems were recovered from 6 patients hospitalized at a university hospital in Gumi, Korea. The isolates were identified with the ATB 32 GN system (bioMérieux, Marcy l’Etoile, France) and 16S–23S rRNA intergenic spacer region sequencing as described previously [9]. Five other A. baumannii strains were involved in this study for comparisons: (i) imipenem-susceptible strain ABA1 carrying the blaOXA-66 gene not preceded by ISAba1; (ii) imipenem-resistant strain ABA2 carrying the blaOXA-23 gene; (iii) imipenem-intermediate strains ABA3 and ABA4 carrying the blaOXA-51-like gene preceded by ISAba1, respectively; and (iv) imipenem-resistant strain ABA5 carrying the blaOXA-66 and blaOXA-23 genes, both of which were preceded by ISAba1.

Antimicrobial susceptibility testing.  Disk diffusion assay was performed using antibiotic-containing disks (Becton-Dickinson, Sparks, MD) for routine antibiograms. The modified Hodge and imipenem and EDTA-sodium mercapto-acetic acid double-disk synergy (IEDDS) tests were conducted on MacConkey agar plates as described previously [10]. MICs of imipenem and meropenem were determined using Etest strips (AB BIODISK, Solna, Sweden) on Mueller-Hinton agar plates with or without a fixed concentration (20 mg/L) of Phe-Arg-β-naphthylamide (PAβN, Sigma-Aldrich, St. Louis, MO) as an efflux pump inhibitor [11].

Gene transfer experiments.  The agar mating method was used to test imipenem resistance transfer using azide-resistant Escherichia coli J53 and rifampin-resistant Pseudomonas aeruginosa PAO 4089 as recipients. Transconjugants were selected on Muller-Hinton agar supplemented with imipenem (0.5 or 2 mg/L) and rifampin (200 mg/L) or azide (100 mg/ L), respectively.

Isoelectric focusing.  Bacterial cell sonicates were used to determine the isoelectric points (pIs) of β-lactamases on a Novex isoelectric focusing (IEF) gel (pH 3–10; Invitrogen, Carlsbad, CA) with a temperature-controlled system (Novel Experimental Technology, San Diego, CA). The pI values were detected by staining the gel with nitrocefin (0.7 mg/L).

SDS-PAGE for OMPs.  Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) was carried out to investigate alterations in OMPs as described previously [10]. Briefly, bacterial cells were disrupted by ultrasonic disintegration and the supernatant was treated with 30% sarkosyl (Sigma-Aldrich). After incubation for 30 min, OMPs were collected by centrifugation at 45,000 x g for 1 hr at 4°C and analyzed by SDS-PAGE on a Mini-PROTEAN 3 Cell apparatus (Bio-Rad, Hercules, CA). The 12.5% (wt/vol) PAGE gels were stained with coomassie brilliant blue.

PCR experiments.  Detection of genes coding the MBLs and OXA carbapenemases was performed by PCR amplification as described previously [12]. Templates for PCR amplification from the clinical isolates were a whole cell lysate. A simple 2-step PCR experiment was conducted for sequencing of the upstream region of the blaOXA-66 gene as described previously [13]. PCR products were subjected to direct sequencing. Both strands of PCR products were sequenced twice with an automatic sequencer (model 3730xl; Applied Biosystems, Weiterstadt, Germany).

Reverse transcriptase-PCR experiments.  Total RNA was extracted from clinical isolates of A. baumannii using RNeasy Protect Bacteria Mini Kit and RNase-free DNase set (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Reverse transcriptase-PCR was performed using Omniscript RT kit (Qiagen) with a random primer (Promega, Madison, WI) and RNase inhibitor (GenDEPOT, Barker, TX) following the manufacturers’ protocols.

Real-time PCR experiments.  The primers and probes employed in real-time PCR experiments using a capillary real-time thermal cycler (LightCycler, Roche Diagnostics, Indianapolis, IN) are listed in Table 1Go. The 5' and 3' ends of probes were labeled with 6-carboxyfluorescein (FAM) and a fluorescence quencher dye (BHQ1), respectively. Amplification was carried out in a 20 µl final volume containing 4 µl of premix (LightCycler Taqman Master kit, Roche Diagnostics), 1 µl of primers (final concentration, 0.5 µM), 0.1 µl of probe (final concentration, 0.1 µM), 9.9 µl of water, and 5 µl of cDNA. The reaction conditions were 95°C for 10 min; 45 cycles of 95°C for 10 sec, 50°C for 30 sec, and 72°C for 1 min. Expression levels of the blaOXA-51-like, adeB, and adeJ genes were normalized against the 16S rRNA. Expression levels of the genes in 5 additional A. baumannii strains were also measured for comparisons (Table 2Go).


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  		Table 1. Nucleotide sequences of primers and probes designed for this study.
 

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  		Table 2. Phenotypic and genotypic characteristics of carbapenem-resistant A. baumannii and control strains.
 
Plasmid analysis.  Plasmids were extracted using a QIAprep Spin Miniprep Kit (Qiagen GmbH) following the manufacturer’s protocol. DNA fragments were separated using 0.8% agarose gels at 50 V/cm for 50 min.

PFGE.  Plugs containing whole genomic DNA of the isolates were digested with SmaI and I-CeuI, respectively. DNA fragments were separated by PFGE using a CHEF-DRII device (Bio-Rad). PFGE conditions of SmaI-macrorestriction analysis were 6 V/cm for 20 hr with pulse times ranging from 0.5 to 60 sec at a temperature of 14°C. Pulse times for I-CeuI restriction analysis were 9 to 90 sec.

Southern blotting.  The gels with plasmids and SmaI- and I-CeuI-digested DNA were blotted onto nylon membrane (Bio-Rad) and hybridized with probe of the blaOXA-23 gene, respectively. The probe was obtained by PCR experiments as described above. Probe labeling, hybridization, and detection were performed with the DIG DNA Labeling and Detection kit (Roche) following the manufacturer’s protocol.

Nucleotide sequence accession numbers.  The nucleotide sequence data reported in this paper are available in the GenBank nucleotide database under accession number FJ360530.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgement
 References
 
Description of A. baumannii isolates.  A. baumannii GM0701 was isolated from a sputum specimen of a 79-yr-old female patient with multiple cerebral infarctions, who was hospitalized in January 2007 on a general medical ward at a university hospital in Gumi, Korea. Disk diffusion assay revealed that the isolate was resistant to all β-lactams including imipenem and meropenem. The isolate also showed resistance to ciprofloxacin, amikacin, gentamicin, tobramycin, and trimethoprimsulfamethoxazole, but it was susceptibile to colistin.

Between March and May 2007, five more clinical isolates of A. baumannii, which showed a similar resistance phenotype to the index case, were recovered from sputum specimens of five patients hospitalized on an intensive care unit of the same hospital. All isolates showed similar SmaI-macrorestriction patterns with <3 band differences by PFGE (Fig. 1Go). All 6 isolates exhibited high-level resistance (>32 mg/L) to imipenem and meropenem by Etest. PAβN at a fixed concentration of 20 mg/L lowered the MICs of meropenem to 4–8 mg/L. All isolates showed positive results on modified Hodge test but they gave negative results on IEDDS test.


Figure 1
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  		Fig. 1. PFGE patterns of genomic DNA of A. baumannii digested by (A) SmaI and (B) I-Ceu1 enzymes. Lanes M, lambda ladder (Bio-Rad) as a DNA size marker.; lanes 1 to 6, experimental isolates; lane N, an imipenem-susceptible clinical isolate of A. baumannii; lane P, an imipenem-resistant clinical isolate of A. baumannii.

 
Location of blaOXA-23  PCR and sequencing experiments detected the blaOXA-23 gene in all 6 isolates, but not genes encoding MBLs, including IMP-1-like, VIM-2-like, and SIM-1-like. The blaOXA-23 gene was preceded upstream by ISAba1. IEF experiments revealed the band of pI 6.7, which corresponds to OXA-23 carbapenemase, in all 6 isolates. The blaOXA-23 gene was not transferred by conjugation to the recipients despite repeated attempts. The blaOXA-23 probe hybridized with 50-kb SmaI and ~500-kb I-CeuI chromosomal fragments, respectively, but not with a plasmid.

Expression of the blaOXA-51-like gene.  Direct sequencing of PCR products showed that all six isolates carried the blaOXA-66 gene. A putative suppressor of F exclusion of phage T7 was found upstream of the blaOXA-66 gene such as A. baumannii ACICU (GenBank No. CP000863 [GenBank] ). However, ISAba1 was not found upstream of the blaOXA-66 gene. Real-time PCR experiments showed that expression levels of the blaOXA-66 gene in these isolates were 20- to 70-times lower than those of strains ABA3, 4, and 5 carrying blaOXA-51-like gene preceded by ISAba1 (Table 2Go).

Expression of OMPs and efflux pumps.  OMPs such as CarO (29 kDa) and 33- to 36- and 43-kDa porins did not show absent or greatly diminished expression in SDS-PAGE. Real-time PCR experiments showed that expression levels of the adeB gene in the six isolates were 10- to 40-times higher than that of strain ABA1 susceptible to carbapenems, while the expression levels of the adeJ gene in these isolates were lowered (Table 2Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgement
 References
 
The six carbapenem-resistant pulmonary isolates of A. baumannii may have originated from the same clone because they showed similar antimicrobial susceptibility and SmaI-macrorestriction patterns. The strain showed positive results on a modified Hodge test but not on the IEDDS test, which suggests that the strain produces carbapenemases but not MBLs. The strain harbored two types of OXA carbapenemases: OXA-23 and OXA-66. OXA-66 was unlikely to play a role in acquiring carbapenem resistance, because ISAba1, which upregulates adjacent bla genes by conferring a promoter, was not found upstream of blaOXA-66. The results of real-time PCR experiments confirmed that the expression levels of blaOXA-66 in the six isolates were 20- to 70-times lower than those of strains carrying the blaOXA-66 gene preceded by ISAba1.

OXA-23 enzyme has been reported to be the dominant carbapenem resistance mechanism in A. baumannii in Asia-Pacific nations [14]. All six isolates carried the blaOXA-23 preceded by ISAba1 containing the gene’s putative promoter (TTAGAA-16 bp-TTATTT) in our study. It was noteworthy that blaOXA-23 gene was identified on chromosomes in all our isolates, while it has mostly been identified on plasmids in previous studies [15].

Lu et al. [16] reported that loss of the CarO OMP causes carbapenem resistance in A. baumannii. However, we could not find any lack or greatly diminished expression of OMPs such as CarO (29 kDa) and 33- to 36- and 43-kDa in our isolates on SDS-PAGE experiments compared to those of the imipenem-susceptible A. baumannii strain. The efflux pump inhibition test using PAβN exhibited prominent reduction of meropenem MICs by 4- to 8-fold, while imipenem MICs showed only slight reduction. Real-time PCR experiments exhibited congruent results since expression levels of the adeB gene in our six isolates were up-regulated. Pannek et al. [17] noted that PAβN has a limited effect on carbapenem MICs in strains that overproduce adeB and the effect is related to the concentration of the inhibitor. However, meropenem MICs for our isolates were lowered by as much as 8-fold at a low concentration (20 mg/L) of PAβN.

We could not confirm that the reduction of meropenem MICs in an efflux pump inhibition test using PAβN was correlated with mRNA levels for the expression of the adeB or adeJ genes. Higgins et al. [18] described that up-regulation of the adeB gene is associated with a decrease in susceptibility to non-fluoroquinolone antibiotics including meropenem. We speculate that overexpression of adeABC efflux pump may play a role in acquiring meropenem resistance in our strains because of the elevated mRNA levels of the adeB gene. Further studies are needed to identify the other efflux pumps that may affect carbapenem resistance.

In conclusion, we suggest that chromosome-borne OXA-23 carbapenemase and overexpression of the AdeABC efflux pump may be associated with carbapenem resistance in our six clinical isolates of A. baumannii.


   Acknowledgement
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
References
 
This work was supported by Korea Food and Drug Administration Grant 08072Hangsaengjae140.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Perez F, Hujer AM, Hujer KM, Decker BK, Rather PN, Bonomo RA. Global challenge of multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2007;51:3471–3484.[Free Full Text]
  2. Munoz-Price LS, Weinstein RA. Acinetobacter infection. NEJM 2008;358:1271–1281.[Medline]
  3. Fournier PE, Richet H. The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis 2006;42:692–699.[Abstract/Free Full Text]
  4. Villegas MV, Kattan JN, Correa A, Lolans K, Guzman AM, Woodford N, Livermore D, Quinn JP. Dissemination of Acinetobacter baumannii clones with OXA-23 carbapenemase in Colombian hospitals. Anti-microb Agents Chemother 2007;51:2001–2004.[Abstract/Free Full Text]
  5. Walther-Rasmussen J, Hoiby N. OXA-type carbapenemases. J Antimicrob Chemother 2006;57:373–383.[Abstract/Free Full Text]
  6. Heritier C, Poirel L, Fournier PE, Claverie JM, Raoult D, Nordmann P. Characterization of the naturally occurring oxacillinase of Acinetobacter baumannii. Antimicrob Agents Chemother 2005;49:4174–4179.[Abstract/Free Full Text]
  7. Heritier C, Poirel L, Lambert T, Nordmann P. Contribution of acquired carbapenem-hydrolyzing oxacillinases to carbapenem resistance in Acinetobacter baumannii. Antimicrob Agents Chemother 2005;49:3198–3202.[Abstract/Free Full Text]
  8. Bratu S, Landman D, Martin DA, Georgescu C, Quale J. Correlation of antimicrobial resistance with beta-lactamases, the OmpA-like porin, and efflux pumps in clinical isolates of Acinetobacter baumannii endemic to New York City. Antimicrob Agents Chemother 2008; 52:2999–3005.[Abstract/Free Full Text]
  9. Chang HC, Wei YF, Dijkshoorn L, Vaneechoutte M, Tang CT, Chang TC. Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S–23S rRNA gene spacer region. J Clin Microbiol 2005;43:1632–1639.[Abstract/Free Full Text]
  10. Lee K, Yong D, Choi YS, Yum JH, Kim JM, Woodford N, Livermore DM, Chong Y. Reduced imipenem susceptibility in Klebsiella pneumoniae clinical isolates with plasmid-mediated CMY-2 and DHA-1 beta-lactamases co-mediated by porin loss. Int J Antimicrob Agents 2007;29:201–206.[Medline]
  11. Ribera A, Ruiz J, Jiminez de Anta MT, Vila J. Effect of an efflux pump inhibitor on the MIC of nalidixic acid for Acinetobacter baumannii and Stenotrophomonas maltophilia clinical isolates. J Antimicrob Chemother 2002;49:697–698.[Free Full Text]
  12. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, Amyes SG, Livermore DM. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2006;27:351–353.[Medline]
  13. Sørensen AB, Duch M, Jorgensen P, Pedersen FS. Amplification and sequence analysis of DNA flanking integrated proviruses by a simple two-step polymerase chain reaction method. J Virol 1993;67:7118–7124.[Abstract/Free Full Text]
  14. Mendes RE, Bell JM, Turnidge JD, Castanheira M, Jones RN. Emergence and widespread dissemination of OXA-23, -24/40 and -58 carbapenemases among Acinetobacter spp. in Asia-Pacific nations: Report from the SENTRY Surveillance Program. J Antimicrob Chemother 2009;63:55–59.[Abstract/Free Full Text]
  15. Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect 2006;12:826–836.[Medline]
  16. Lu PL, Doumith M, Livermore DM, Chen TP, Woodford N. Diversity of carbapenem resistance mechanisms in Acinetobacter baumannii from a Taiwan hospital: spread of plasmid-borne OXA-72 carbapenemase. J Antimicrob Chemother 2009;63:641–647.[Abstract/Free Full Text]
  17. Pannek S, Higgins PG, Steinke P, Jonas D, Akova M, Bohnert JA, Seifert H, Kern WV. Multidrug efflux inhibition in Acinetobacter baumannii: comparison between 1-(1-naphthylmethyl)-piperazine and phenyl-arginine-beta-naphthylamide. J Antimicrob Chemother 2006;57:970–974.[Abstract/Free Full Text]
  18. Higgins PG, Wisplinghoff H, Stefanik D, Seifert H. Selection of topoisomerase mutations and overexpression of adeB mRNA transcripts during an outbreak of Acinetobacter baumannii. J Antimicrob Chemother 2004;54:821–823.[Abstract/Free Full Text]



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