Novel gyrA and parC point mutations in two strains of Acinetobacter baumannii resistant to ciprofloxacin

Ahmed Hamouda and Sebastian G. B. Amyes*

Division of Medical Microbiology, School of Biomedical and Clinical Laboratory Sciences, Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK

Keywords: A. baumannii , fluoroquinolones , resistance

Sir,

Acinetobacter baumannii are now resistant to most antibacterial drugs, with some centres reporting up to 80% of strains resistant to all aminoglycosides, and there is resistance to imipenem and sulbactam. In addition, resistance to ciprofloxacin has rapidly emerged in clinical isolates.1 Fluoroquinolone resistance in A. baumannii has been associated with mutations in the gyrA and parC genes.2,3 It has been found that the amino acid residues most frequently mutated in GyrA from A. baumannii occur at Ser-83 and Gly-81.2 Ser-80 and Glu-84 were hotspots for mutation within ParC.3 The purpose of this study was to screen Scottish A. baumannii clinical isolates for these mutations and/or new mutations associated with ciprofloxacin resistance. We examined nine epidemiologically unrelated ciprofloxacin-resistant clinical isolates of A. baumannii, using pulsed-field gel electrophoresis, collected from five Scottish hospitals: Aberdeen Royal Infirmary, Aberdeen; Raigmore Hospital, Inverness; Ninewells Hospital, Dundee; Western General Hospital and Royal Infirmary both in Edinburgh. The MIC of ciprofloxacin was determined by the agar double-dilution method following the British Society for Antimicrobial Chemotherapy guidelines.4 All isolates were identified as resistant to ciprofloxacin (MIC ≥4 mg/L). The primers used for the amplification of the quinolone resistance determining region of gyrA were the same as those used by Vila et al.2 However, the primers used for parC were designed with Primer3 Imput software http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi, and were as follows: 5'-AAAAATCAGCGCGTACAGTG-3' and 5'-CGAGAGTTTGGCTTCGGTAT-3'. All isolates but one had changes affecting GyrA: eight of Ser-83->Leu and one of Gly-81->Cys. Two highly ciprofloxacin-resistant isolates (MICs of ciprofloxacin, 64 mg/L) had a single mutation in ParC, either Glu-84->Lys or Gly-78->Cys. The mutations in GyrA and ParC are shown in Table 1. Vila et al.2,3 found that only two, of 21 clinical isolates analysed, had Gly-81 and Ala-84 residue changes in GyrA and suggested that these mutations contribute little, if at all, to ciprofloxacin resistance. In contrast, we found that changes at codon 81 conferred a ciprofloxacin-resistance level of 4 mg/L where glycine had been substituted by cysteine with no mutation concurrent at codon Ser-83. This mutation has already been described in Escherichia coli.5 The GyrA mutations do not explain why isolates with the same mutation have different MICs of ciprofloxacin. This variation in MICs could be explained by a mutation in the parC gene. In A. baumannii, topoisomerase IV is a target of quinolones and mutation at residues Ser-80 and Glu-84 of ParC contribute to decreased fluoroquinolone susceptibility.3 In the present study, only a change at residue Glu-84 was observed. This study showed that a double mutation in both gyrA and parC genes is needed to acquire a high-level resistance to ciprofloxacin and this is similar to previously published data.2,3 In contrast, in E. coli a double mutation affecting Ser-83 of GyrA and Ser-80 of ParC leads only to a moderate level of resistance to ciprofloxacin, whereas three or four mutations in both gyrA and parC genes are required to obtain high-level resistance. This difference is probably because of the decreased permeability of A. baumannii to quinolones.6 Moreover, no parC mutation has been found without the presence of mutation in the gyrA gene, suggesting that parC could be a secondary target for quinolones.


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Table 1. Quinolone resistance mutations in GyrA and ParC of ciprofloxacin-resistant clinical isolates of A. baumannii

 
In A. baumannii, restriction fragment length polymorphism was used to detect mutations at codon 83 in gyrA or at codon 80 in parC.2,3 The presence or absence of mutations in these genes at these particular codons can be determined by the digestion of PCR products with HinfI. HinfI restriction of the PCR products of gyrA from isolate 8 (MIC = 4 mg/L) and a ciprofloxacin-susceptible strain yielded two fragments of 291 bp and 52 bp, indicating that the HinfI site was intact. For the rest of the isolates the restriction failed to produce restriction fragments, indicating the loss of the HinfI site at the codons for amino acids Asp-82–Ser-83, and suggesting that mutation either at codon Ser-82 or Ser-83 had occurred. HinfI restriction of PCR products of parC produced two DNA fragments of 144 bp and 53 bp, respectively, in all isolates, indicating that the HinfI restriction site at the codons for amino acids Asp-79–Ser-80 was intact, and suggesting the absence of mutation at codon Ser-80.

In conclusion, we showed that mutations in the amino acids corresponding to Gly-81 and Gly-78 in GyrA and ParC, respectively, contribute to decreased ciprofloxacin susceptibility in A. baumannii. Furthermore, it is also possible that other mutations at other locations within gyrA, parC, or in other genes may also contribute to the modulation of the MIC level since these mutations did not entirely explain resistance.

Acknowledgements

We gratefully acknowledge the University of Edinburgh for supporting this work.

Footnotes

* Corresponding author. Tel: +44-131-650-3163; Fax: +44-131-650-6882; Email: s.g.b.amyes{at}ed.ac.uk

References

1 . Acar, J. F., O'Brien, T. F., Goldstein, F. W. et al. (1993). The epidemiology of bacterial resistance to quinolones. Drugs 45, Suppl. 3, 24–8.

2 . Vila, J., Ruiz, J., Goni, P. et al. (1995). Mutation in the gyrA gene of quinolone-resistant clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 39, 1201–3.[Abstract]

3 . 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]

4 . British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

5 . Yoshida, H., Bogaki, M., Nakamura, M. et al. (1990). Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrobial Agents and Chemotherapy 34, 1271–2.[ISI][Medline]

6 . Sato, K. & Nakae, T. (1991). Outer membrane permeability of Acinetobacter calcoaceticus and its implication in antibiotic resistance. Journal of Antimicrobial Chemotherapy 28, 35–45.[Abstract]





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