Correspondence laboratories, indicating further spread of strain A since its original description.1 To our knowledge, this is also the first report of group 2 CTX-M enzymes in the UK; the isolates were from three hospitals and it is possible that they represent separate introductions. Group 2 are the most prevalent CTX-M enzymes in parts of South America and in Israel;3 group 9 enzymes are prevalent in Spain.5 In summary, we have developed a multiplex PCR assay that was able to detect and distinguish alleles encoding CTX-M enzymes belonging to all five phylogenetic groups. CTX-M ESBLs are recognized worldwide as an increasingly serious public health concern.3 Simple PCR assays, as described here and by others,6 will assist in monitoring their emergence and dissemination.
Acknowledgements We wish to thank Vicky Ensor, Peter Hawkey, Katie Hopkins and Patrice Nordmann for providing some of the control strains included in this study. Part of this work was presented at the 15th ECCMID, Copenhagen, April 2005.
Transparency declarations No declarations were made by the authors of this paper.
References 1. Woodford N, Ward ME, Kaufmann ME et al. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum b-lactamases in the UK. J Antimicrob Chemother 2004; 54: 735–43. 2. Munday CJ, Whitehead GM, Todd NJ et al. Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum b-lactamases in York, UK. J Antimicrob Chemother 2004; 54: 628–33. 3. Bonnet R. Growing group of extended-spectrum b-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: 1–14. 4. Saladin M, Cao VT, Lambert T et al. Diversity of CTX-M b-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 2002; 209: 161–8. 5. Hernandez JR, Martinez-Martinez L, Canton R et al. Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum b-lactamases in Spain. Antimicrob Agents Chemother 2005; 49: 2122–5. 6. Pitout JDD, Hossain A, Hanson ND. Phenotypic and molecular detection of CTX-M-b-lactamases produced by Escherichia coli and Klebsiella spp. J Clin Microbiol 2004; 42: 5715–21.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dki397 Advance Access publication 15 November 2005
Development of highly ciprofloxacin-resistant laboratory mutants of Acinetobacter baumannii lacking topoisomerase IV gene mutations A. Hamouda and S. G. B. Amyes* Molecular Chemotherapy, Centre for Infectious Diseases, The Chancellor’s Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, UK
Keywords: A. baumannii, parC, outer membrane proteins, quinolones, resistance *Corresponding author. Tel: +44-131-242-6652; Fax: +44-131242-6611; E-mail:
[email protected] 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 ciprofloxacinresistant 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: 50 -AATCTGCCCGTGTCGTTGGT-30 and 50 -GCCATACCTACGGCGATACC-30 for gyrA, and 50 -AAAAATCAGCGCGTACAGTG-30 and 50 -CGAGAGTTTGGCTTCGGTAT-30 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 Ser83!Leu (C!T 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
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Correspondence Table 1. Mutations in gyrA and parC genes and MIC values for ciprofloxacin-resistant mutants of A. baumannii MIC (mg/L)
Amino acid substitutions
Strain
CIP
IPM
TET
TMP
GEN
CHL
CTX
gyrA
ATCC 19606a C1 C2 C3 C4
1 8 32 64 128
0.06 0.12 0.12 0.12 0.12
2 2 2 4 4
4 4 8 32 32
1 2 2 4 8
128 256 256 256 256
8 8 32 32 32
no mutation Ser-83!Leu Ser-83!Leu Ser-83!Leu Ser-83!Leu
parC no no no no no
OMP 20 kDa band
mutation mutation mutation mutation mutation
+ – – – –
OMP, outer membrane protein; CIP, ciprofloxacin; IPM, imipenem; TET, tetracycline; TMP, trimethoprim; GEN, gentamicin; CHL, chloramphenicol; CTX, cefotaxime, +, 20 kDa band conserved; –, 20 kDa band loss. Ser, serine; Leu, leucine; C1, ciprofloxacin first step mutant; C2, ciprofloxacin second step mutant; C3, ciprofloxacin third step mutant; C4, ciprofloxacin forth step mutant. a Wild-type.
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.
Acknowledgements This study was supported by Altajir World of Islam Trust.
Transparency declarations No declarations were made by the authors of this paper.
References 1. Vila J, Ruiz J, Goni P et al. Mutation in the gyrA gene of quinoloneresistant 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.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dki408 Advance Access publication 10 November 2005
Occurrence of extended-spectrum b-lactamases and plasmid-mediated AmpC b-lactamases among Korean isolates of Proteus mirabilis Yeon-Joon Park1*, Seungok Lee2, Yang Ree Eun-Jee Oh1, Gun-Jo Woo4 and Kyungwon Lee5 1
Kim3,
Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Kangnam St Mary’s Hospital, 505 Banpo-dong, Seocho-ku, Seoul, 137-701, Korea; 2Seoul Medical Science Institute, Seoul Clinical Laboratories, Seoul, Korea; 3Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea; 4Korea Food and Drug Administration, Seoul, Korea; 5Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea
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