Selection of topoisomerase mutations and overexpression of adeB mRNA transcripts during an outbreak of Acinetobacter baumannii

Paul G. Higgins*, Hilmar Wisplinghoff, Danuta Stefanik and Harald Seifert

Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19–21, 50935 Cologne, Germany

Received 28 June 2004; returned 30 July 2004; revised 6 August 2004; accepted 11 August 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: To investigate the mechanism of ciprofloxacin resistance in isolates of Acinetobacter baumannii during two hospital outbreaks and to determine the expression level of the gene encoding the AdeB efflux pump.

Methods: Isolates were previously typed by PFGE and their MICs determined by broth microdilution. The gyrA and parC genes were sequenced and the adeB gene examined by real-time reverse transcription PCR (RT-PCR).

Results: Two clonal lineages were responsible for the two hospital outbreaks. In both outbreaks, ciprofloxacin susceptibility was reduced during the course of the outbreak when compared with the index isolates. Mutations in gyrA and parC were found to have occurred during the outbreak. The MICs of non-fluoroquinolone antibiotics were raised in one clonal lineage and this was associated with a >10-fold increase in mRNA transcripts for adeB.

Conclusions: We have witnessed the appearance of gyrA and parC mutations during outbreaks of A. baumannii. In parallel with these mutations, we observed up-regulation of the adeB gene associated with a decrease in susceptibility to non-fluoroquinolone antibiotics. These data illustrate the propensity for A. baumannii to develop multi-drug resistance rapidly.

Keywords: ciprofloxacin , efflux , RT-PCR , multidrug resistance


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Acinetobacter baumannii is increasingly being isolated in the hospital setting, where it is a cause of serious morbidity and mortality in the immunocompromised patient. Of even greater concern is the clonal spread of strains that are increasingly hard to eradicate due to their multi-drug resistance, tolerance of commonly used disinfectants and their ability to survive long periods on dry surfaces.1,2

Fluoroquinolones are very potent antimicrobials that are sometimes used liberally in the hospital setting. The blanket use of fluoroquinolones is one cause of the emergence of non-traditional pathogens, such as A. baumannii, which are selected when other more susceptible organisms are removed.3 Indeed, risk factors for A. baumannii infection include the prior use of intravenous ciprofloxacin.4 Resistance to fluoroquinolones is mediated primarily through spontaneous mutations in the targets of these drugs, DNA gyrase and topoisomerase IV. A secondary route to fluoroquinolone resistance is through active efflux of the drug. Efflux pumps typically have a broad substrate specificity that can lead to resistance against several unrelated drug classes. In A. baumannii, one such efflux pump has been described, AdeB.5 This pump has been shown to be responsible for aminoglycoside resistance and is involved in reduced susceptibility to fluoroquinolones.

In the present study, we report on the appearance of gyrA and parC mutations in A. baumannii clonal strains isolated during two separate hospital outbreaks, and the selection of clones that have become resistant to non-fluoroquinolone antibiotics associated with up-regulation of the efflux pump AdeB.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial strains and antibiotic susceptibility testing

Clonal A. baumannii isolates were part of a previous study and chosen on the basis of increasing resistance to ciprofloxacin in two outbreaks that occurred in 1991.6 The index G-II strain (U10247) was isolated from a patient's urine sample on 15 November 1991. Two further isolates with nearly identical pulsed-field gel electrophoresis patterns (one band difference) and increased resistance to ciprofloxacin were isolated on 13 and 20 December 1991 from separate patients (U11177 and U11432, respectively) in the same rehabilitation unit. Altogether, nine patients were involved in the hospital outbreak over a period of 6 months. The G-IV index strain (St16706) was isolated from a blood culture on 7 September 1991, and a ciprofloxacin-resistant strain was isolated on 10 November 1991 (St20820) from the same burns unit. The outbreak extended over 16 months and involved 34 patients. MICs were determined by broth microdilution according to the NCCLS guidelines for susceptibility testing, and the results were interpreted according to NCCLS criteria.7 The standard A. baumannii ATCC 19606 was used as the quality control strain for susceptibility studies, gyrA and parC sequence determination. Escherichia coli ATCC 25922 was used as a control for real-time reverse transcription PCR (RT-PCR).

Characterization of gyrA and parC quinolone resistance-determining region (QRDR)

Primers (MWG Biotech, Ebersberg, Germany) and conditions for PCR of the gyrA and parC QRDR were as previously described.8 PCR products were sequenced using the same primers.

RT-PCR

Cells were grown aerobically in LB broth until mid-log phase; aliquots were removed and immediately added to RNAprotect (Qiagen, Hilden, Germany). Total cellular RNA was extracted using the Qiagen RNeasy columns, and contaminating DNA was removed with DNaseI on-column digestion. RNA was examined for integrity by denaturing gel electrophoresis and its concentration quantified in a spectrophotometer. Equal quantities of total RNA (1 µg) were reverse transcribed into cDNA using Omniscript reverse transcriptase (Qiagen) and 10 µM random hexamers (MWG Biotech). RT-PCR was performed in a LightCycler (Roche, Mannheim, Germany) with QuantiTect SYBR Green (Qiagen) to detect transcripts of adeB. 16sRNA was used as a housekeeping gene to normalize levels of adeB transcripts. Expression levels were compared against those of the first clones isolated and expressed as a percentage. Control RT-PCR reactions of untranscribed RNA were performed in tandem. Primer sequences for adeB and 16sRNA were: adeB forward, 5'-GGATTATGGCGACAGAAGGA-3'; adeB reverse, 5'-AATACTGCCGCCAATACCAG-3'; 16sRNA forward, 5'-CAGCTCGTGTCGTGAGATGT- 3'; 16sRNA reverse, 5'-CGTAAGGGCCATGATGACTT- 3'. Real-Time Cycling parameters for adeB were: one cycle of 95°C 15 min followed by 40 cycles of 94°C 15 s, 54°C 20 s, 72°C 20 s. For 16sRNA, the annealing temperature was 55°C for 30 cycles. A melting-curve analysis was performed post-PCR under the following conditions: temperature ramp to 95°C for 0 s, cooling to 65°C for 15 s at a slope of 20°C/s followed by a 0.1°C/s rise in temperature up to 95°C.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
In this study, we have analysed the mechanism of fluoroquinolone resistance in five A. baumannii isolates from two separate outbreaks collected during the winter of 1991 by investigating mutations in the gyrA and parC genes and expression of adeB.6 MIC data for these isolates are presented in Table 1. U10247, the index strain for the GII outbreak, was resistant to ciprofloxacin and at breakpoint for ofloxacin, according to NCCLS guidelines. U11177 and U11432 were recovered later and showed high-level ciprofloxacin and ofloxacin resistance. This highly resistant strain became the established outbreak strain and replaced the less susceptible phenotype, which was no longer recovered. In a separate outbreak, the G-IV index strain St16706 was susceptible to ciprofloxacin and ofloxacin, but had become resistant to ciprofloxacin and intermediate against ofloxacin when recovered at a later date in a different patient on the same ward (St20820). In both outbreaks, the sparfloxacin MIC was also raised. The results of sequencing the gyrA and parC genes are shown in Table 1. The index strain from G-II initially had a Ser-83->Leu substitution in GyrA. This substitution is associated with low-level ciprofloxacin resistance.8 The two strains recovered during the course of the G-II outbreak had an additional Ser-80->Leu substitution in ParC. It is of particular interest to note that strains that have mutated in parC would have been selected by a fluoroquinolone at a subinhibitory concentration. When these strains were isolated, ciprofloxacin was the only approved fluoroquinolone used in these hospitals and its pharmacokinetics would not have allowed a concentration >32 mg/L to be achieved.


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Table 1. MICs and translated sequences of gyrA and parC

 
St16706 was wild-type in both GyrA and ParC; however, in St20820 a Ser-83->Leu substitution has appeared in GyrA conferring resistance to ciprofloxacin. The appearance of gyrA and parC mutations in vivo has previously been demonstrated with Streptococcus pneumoniae and Pseudomonas aeruginosa.9,10

MICs of non-fluoroquinolone antibiotics were found to have risen in the G-II strains (Table 1). The G-II clones U11177 and U11432 showed reduced susceptibility to meropenem, became resistant to netilmicin and tetracycline and showed increased resistance to tobramycin and clarithromycin. This rise in MIC suggests the involvement of enhanced drug efflux. Magnet et al.5 have previously shown that AdeB can confer resistance to aminoglycosides and reduce susceptibility to macrolides, fluoroquinolones and tetracylines. Investigation into expression of the adeB gene revealed the pump to be expressed constitutively in the A. baumannii clones, but was not detected in E. coli ATCC 25922. We found a 20-fold increase in mRNA transcripts from U11177 and U11432 compared with U10247 (Figure 1). Up-regulation of the P. aeruginosa efflux pumps MexCD-OprJ and MexEF-OprN has been selected in P. aeruginosa during ciprofloxacin therapy in cystic fibrosis patients and was the predominant mechanism of fluoroquinolone resistance.10 However, whereas the development of efflux mutations took place over a 3 year period in P. aeruginosa, our isolates mutated in a matter of weeks. There was no difference in non-fluoroquinolone MICs with G-IV strains and we found no evidence for increased expression of adeB mRNA in St20820.



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Figure 1. Expression of adeB mRNA transcripts in the clinical isolates. *No transcripts detected.

 
In summary, A. baumannii has been shown to mutate rapidly in gyrA and parC and at the same time to up-regulate the efflux pump AdeB. The up-regulation of efflux transcripts was associated with a multidrug-resistant phenotype. Although we have no detailed patient data, it is likely that ciprofloxacin pressure was responsible for the selection of the efflux phenotype in addition to the topoisomerase mutations. In both outbreaks, the strains showing the lower MICs were not recovered from more than one patient, whereas the strains exhibiting the higher fluoroquinolone MICs became the outbreak strain that was transferred to multiple patients. It therefore appears that it was the increased ciprofloxacin resistance that contributed to the strains' epidemicity. This underlines the statement by Paterson3 that fluoroquinolones are ‘not suitable for sustained use in hospitals as "workhorse" antibiotic therapy’.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We would like to thank the Koeln Fortune Program/Faculty of Medicine, University of Cologne for supporting P. G. H.


    Footnotes
 
* Corresponding author. Tel: +49-221-4783071; Fax: +49-221-4783067; Email: paul.higgins{at}uni-koeln.de


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Jawad, A., Seifert, H., Snelling, A. M. et al. (1998). Survival of Acinetobacter baumannii on dry surfaces: Comparison of outbreak and sporadic isolates. Journal of Clinical Microbiology 36, 1938–41.[Abstract/Free Full Text]

2 . Koeleman, J. G. M., van der Bijl, M. W., Stoof, J. et al. (2001). Antibiotic resistance is a major risk factor for epidemic behavior of Acinetobacter baumannii. Infection Control and Hospital Epidemiology 22, 284–8.[ISI][Medline]

3 . Paterson, D. L. (2004). "Collateral damage" from cephalosporin or quinolone antibiotic therapy. Clinical Infectious Diseases 38, S341–5.[CrossRef][ISI][Medline]

4 . Villers, D., Espaze, E., Coste Burel, M. et al. (1998). Nosocomial Acinetobacter baumannii infections: microbiological and clinical epidemiology. Annals of Internal Medicine 129, 182–9.[Abstract/Free Full Text]

5 . Magnet, S., Courvalin, P. & Lambert, T. (2001). Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrobial Agents and Chemotherapy 45, 3375–80.[Abstract/Free Full Text]

6 . Seifert, H. & Gerner Smidt, P. (1995). Comparison of ribotyping and pulsed-field gel-electrophoresis for molecular typing of Acinetobacter isolates. Journal of Clinical Microbiology 33, 1402–7.[Abstract]

7 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

8 . Vila, J., Ruiz, J., Goni, P. et al. (1997). Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy 39, 757–62.[Abstract]

9 . Davidson, R., Cavalcanti, R., Brunton, J. L. et al. (2002). Brief report: resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. New England Journal of Medicine 346, 747–50.[Free Full Text]

10 . Jalal, S., Ciofu, O., Hoiby, N. et al. (2000). Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrobial Agents and Chemotherapy 44, 710–2.[Abstract/Free Full Text]