JAC Advance Access originally published online on November 15, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):155-156; doi:10.1093/jac/dki397
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Correspondence |
Development of highly ciprofloxacin-resistant laboratory mutants of Acinetobacter baumannii lacking topoisomerase IV gene mutations
Molecular Chemotherapy, Centre for Infectious Diseases, The Chancellor's Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK
* Corresponding author. Tel: +44-131-242-6652; Fax: +44-131-242-6611; E-mail: S.G.B.Amyes{at}ed.ac.uk
Keywords: A. baumannii , parC , outer membrane proteins , quinolones , resistance
Sir,
Mutations in topoisomerase genes associated with decreased susceptibility to ciprofloxacin have been described in clinical isolates of Acinetobacter baumannii.1,2 It has been suggested that ParC is a secondary target for quinolones which renders this species highly resistant to these drugs.2 In addition, alterations in cellular permeability or active drug efflux may contribute to quinolone resistance in A. baumannii.2 The purpose of this study was to analyse the contribution of topoisomerase IV mutations and drug permeation to high-level ciprofloxacin resistance in A. baumannii laboratory mutants. Spontaneous ciprofloxacin-resistant mutants were selected by exposing A. baumannii ATCC 19606 to increasing concentrations of ciprofloxacin (1–4 times the MIC). This process was repeated until the MICs of ciprofloxacin were 128 mg/L. The MIC was determined using the agar double-dilution method following the guidelines of the British Society for Antimicrobial Chemotherapy.3 The quinolone resistance determining regions (QRDRs) of gyrA and parC were amplified with the following primer pairs: 5'-AATCTGCCCGTGTCGTTGGT-3' and 5'-GCCATACCTACGGCGATACC-3' for gyrA, and 5'-AAAAATCAGCGCGTACAGTG-3' and 5'-CGAGAGTTTGGCTTCGGTAT-3' for parC.
The primers used for parC were designed using the primer software at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi (15 January 2004, date last accessed). PCR primers were also used as sequencing primers. The gyrA and parC sequences were analysed by the Blast online search facility at http://www.ncbi.nlm.nih.gov/BLAST/ (30 May 2005, date last accessed) and compared with those of the quinolone-susceptible parent strain. Four consecutive selective steps were needed for the development of ciprofloxacin-resistant mutants (breakpoint 
4 mg/L) and we defined isolates with an MIC of ciprofloxacin of 32 mg/L as highly resistant. The susceptibilities to ciprofloxacin and other non-related antibiotics of mutants are shown in Table 1. There was an increase (8- to 128-fold) in the MICs of ciprofloxacin required to inhibit the growth of mutants compared with that of the wild-type. Resistant mutants were used for further investigation by PCR amplification and DNA sequencing of the QRDRs of the gyrA and parC genes. Amplification of gyrA and parC yielded PCR products of 343 and 197 bp, respectively. The sequences of the QRDR of gyrA showed a change of Ser-83
Leu (CT transversion from codon TCA) in all mutants but no mutations were found in the parC gene (Table 1). The outer membrane proteins of four mutants [C1 (ciprofloxacin, MIC = 8 mg/L), C2 (MIC = 32 mg/L), C3 (MIC = 64 mg/L) and C4 (MIC = 128 mg/L)] were investigated by SDS–PAGE. Outer membrane profiles from mutants were compared with that of the A. baumannii ATCC 19606 susceptible strain. The major band of 40 kDa was conserved in all mutants, however, there was a deletion of a 20 kDa band. Spencer and Towner4 examined clinical isolates of A. baumannii resistant to ciprofloxacin and susceptible to moxifloxacin for their ability to generate spontaneous moxifloxacin-resistant isolates. They found that ciprofloxacin-resistant isolates failed to produce stable moxifloxacin-resistant mutants at detectable frequencies. In contrast, in the present study, ciprofloxacin-resistant mutants were stable when plated 10 times on ciprofloxacin-free agar and retained their resistance to ciprofloxacin. A gyrA mutation was found in all resistant mutants where serine is substituted by leucine at position 83. This mutation seems to be the most frequently found in clinical and laboratory quinolone-resistant isolates of many Gram-negative bacteria including A. baumannii.1 Also, it appears to be a prerequisite for further mechanism(s) of resistance. However, no parC mutations at codons 80 or 84 were found in mutants with ciprofloxacin MICs 32 mg/L, a value at which such mutations may occur.2 Moreover, in contrast to gyrA, these parC point mutations seem to be difficult to select in vivo, which begs the question of whether mutations within topoisomerase IV can only be selected in vivo and only under certain conditions. An in vivo experiment would be a good approach to solve this problem. As mutation within gyrA could not explain the MIC changes seen alone the role of permeability was investigated. The loss of a 20 kDa band in all isolates is an interesting observation, but this was observed at all four stages in the mutation process (Table 1). Investigation of the function of this protein may reveal its involvement in ciprofloxacin resistance. Hooper et al.5 showed that alterations in the DNA gyrA subunit and the OmpF outer membrane porin protein were the main mechanisms of resistance in a norfloxacin-resistant mutant. The phenotypic results suggest that the activation of an efflux system may be responsible for the antibiotic resistance seen in the mutants (Table 1). The AdeB efflux pump has previously been linked to aminoglycoside resistance in A. baumannii.6
|
In conclusion, our findings highlight that ParC was not a secondary target for quinolones in A. baumannii laboratory mutants resistant to ciprofloxacin. In addition, with the loss of a 20 kDa band, it is possible that permeability is an alternative pathway of resistance to fluoroquinolones, at least in these mutants as no additional gyrA or parC mutations were present.
Transparency declarations
No declarations were made by the authors of this paper.
Acknowledgements
This study was supported by Altajir World of Islam Trust.
References
1. Vila J, Ruiz J, Goni P et al. Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. J Antimicrob Chemother 1995; 39: 1201–3.
2. Vila J, Ruiz J, Goni P et al. Quinolone-resistance mutations in topoisomerase IV parC gene of Acinetobacter baumannii. Antimicrob Agents Chemother 1997; 39: 757–62.
3.
British Society for Antimicrobial Chemotherapy. A guide to sensitivity testing. J Antimicrob Chemother 1991; 27 Suppl D: 1–50.
4.
Spencer RP, Towner KJ. Frequencies and mechanisms of resistance to moxifloxacin in nosocomial isolates of Acinetobacter baumannii. J Antimicrob Chemother 2003; 52: 687–90.
5.
Hooper DC, Wolfson JS, Souza KS et al. Genetic and biochemical characterization of norfloxacin resistance in Escherichia coli. Antimicrob Agents Chemother 1986; 29: 639–44.
6.
Magnet S, Courvalin P, Lambert T. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob Agents Chemother 2001; 45: 3375–80.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||