JAC Advance Access originally published online on August 10, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):905-907; doi:10.1093/jac/dkl324
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Correspondence |
Evolution of extended-spectrum ß-lactamases in a MutS-deficient Pseudomonas aeruginosa hypermutator
Antimicrobial Research Centre and Research Institute of Molecular and Cellular Biology, University of Leeds Leeds LS2 9JT, UK
*Corresponding author. Tel: +44-113-343-5604; Fax: +44-113-343-3167; E-mail: i.chopra{at}leeds.ac.uk
Keywords: resistance , ceftazidime , mutation
Sir,
Bacteria exhibiting elevated mutation frequencies (hypermutators), often through deficiencies in the methyl directed mismatch repair (MMR) pathway, have been reported in clinical isolates of several pathogenic species and there is growing awareness of the importance of these organisms in the emergence and spread of antibiotic resistance in bacteria.
In Gram-negative bacteria, extended-spectrum ß-lactamases (ESBLs) derived by mutation from TEM or SHV (class A) and OXA (class D) enzymes have arisen with the ability to hydrolyse third-generation cephalosporins. Since ESBLs evolve by acquisition of point mutations that enlarge the enzyme active site, hypermutable Gram-negative bacteria could represent hosts where these evolutionary events occur more rapidly than in strains with normal mutation frequencies, as shown recently in Escherichia coli.1
Since hypermutable strains of Pseudomonas aeruginosa occur in the clinical setting, their superior mutation activities could facilitate ESBL evolution.2 The aim of the present study was to investigate the development of ESBLs in a MutS-deficient P. aeruginosa strain by examining whether clinically important TEM-2- and OXA-10-derived ESBLs would evolve in a hypermutable derivative of P. aeruginosa PAO1.
P. aeruginosa PAO1 and PAO
mutS strains were provided by Dr R. Canton, Ramon y Cajal University Hospital, Madrid, Spain.3 The naturally occurring plasmids RP4 encoding TEM-2 (Kmr, Tcr, bla-TEM2) and R151 encoding OXA-10 (Genr, Kmr, Sur, bla-OXA10) were obtained respectively from Professor P. M. Bennett, University of Bristol, and Dr D. M. Livermore, Centre for Infections, Health Protection Agency, London.
MICs were determined by 2-fold serial dilution in Iso-Sensitest agar and the MIC was defined as the lowest antibiotic concentration that completely inhibited growth.
To generate ceftazidime-resistant mutants, strains were serially subcultured in Iso-Sensitest broth overnight at 37°C containing doubling concentrations of ceftazidime from 16 to 512 mg/L. A number of the resulting mutants were characterized by sequencing the blaTEM-2 and blaOXA-10 genes using primers and conditions described by Danel et al.4
To determine whether resistance was plasmid encoded, plasmids were transferred by conjugation from mutants to a spontaneously generated rifampicin-resistant mutant of P. aeruginosa PAOmutS and the resulting transconjugants were tested for susceptibility to ceftazidime. The presence of plasmid R151 in the transconjugants was confirmed by resistance to streptomycin. The presence of RP4 in the transconjugants was confirmed by PCR amplification of the genes encoding the essential transfer protein TraF using primers 5'-GCAAGCAGCCGCTCTACTTC and 5'-TCGGAGCCGCATTCGTAA and the partitioning system protein ParD using primers 5'-CCGTTTAAGGACATGAGCGAACTCT and 5'-CAGCGCAATGGTGGTGGCTAGTCTG.
We confirmed the increased mutation frequency of PAOmutS reported by Oliver et al.3 (data not shown). The MICs of ceftazidime for mutants generated by serial subculture are given in Table 1. Transconjugants containing the RP4-derived plasmids displayed similar ceftazidime resistance profiles to the original mutants (Table 1), indicating that in each case resistance was plasmid-mediated. However, transconjugants derived by transfer of R151 from the original ceftazidime-resistant mutants displayed lower levels of resistance to ceftazidime than the starting strains (Table 1), indicating that although R151 encoded ß-lactamases contributed to ceftazidime resistance in the primary mutants, additional mechanisms may have been selected. Nevertheless, the ceftazidime MICs against the transconjugants are consistent with the selection of one or more OXA-10-derived ESBLs in P. aeruginosa PAOmutS (R151).
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Three amino acid substitutions were identified in the TEM mutants, which corresponded to TEM-11, TEM-61 and a third novel enzyme. Both TEM-11 and TEM-61 were originally isolated from a patient undergoing ceftazidime treatment for a Klebsiella pneumoniae infection.5 The mutation I173T identified in mutant 512-T4 has not been previously reported; substitution at this position only occurs in TEM-132, but involves a valine for isoleucine alteration in the naturally occurring enzyme.6 A single amino acid substitution not corresponding to any naturally occurring ß-lactamase was identified in the mutants derived from OXA-10 (Table 1). Danel et al.4 also generated (with non-hypermutable P. aeruginosa) an OXA-derived ESBL from R151 that does not correspond to a naturally occurring enzyme.
Although ceftazidime resistance has been associated with hypermutability in clinical isolates of P. aeruginosa from chronic lung infections2 it is not known whether these strains contain ESBLs. Indeed on the basis of data reported here, we suggest that hypermutable P. aeruginosa may not be the source of those ESBLs found in clinical isolates of this organism. This situation may reflect the relative ease by which derepression of AmpC in P. aeruginosa provides a route for resistance to expanded-spectrum ß-lactams.
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References
1 Ellington MJ, Livermore DM, Pitt TL, et al. (2005) Accelerated development of TEM-29 extended-spectrum ß-lactamase from TEM-1 in hypermutable, mutS, Escherichia coli. Programs and Abstracts of the Fifteenth European Congress of Clinical Microbiology and Infectious DiseasesCopenhagen, DenmarkClin Microbiol Infect 11:Suppl 2, pp. 123 2005. Abstract P475.
2
Macia MD, Blanquer D, Togores B, et al. (2005) Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections. Antimicrob Agents Chemother 49:3382–6.
3
Oliver A, Levin BR, Juan C, et al. (2004) Hypermutation and the preexistence of antibiotic-resistant Pseudomonas aeruginosa mutants: implications for susceptibility testing and treatment of chronic infections. Antimicrob Agents Chemother 48:4226–33.
4
Danel F, Hall LM, Livermore DM. (1999) Laboratory mutants of OXA-10 ß-lactamase giving ceftazidime resistance in Pseudomonas aeruginosa. J Antimicrob Chemother 43:339–44.
5
Vuye A, Verschraegen G, Claeys G. (1989) Plasmid-mediated ß-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli resistant to ceftazidime. Antimicrob Agents Chemother 33:757–61.
6
Zarnayova M, Siebor E, Pechinot A, et al. (2005) Survey of Enterobacteriaceae producing extended-spectrum ß-lactamases in a Slovak hospital: dominance of SHV-2a and characterization of TEM-132. Antimicrob Agents Chemother 49:3066–9.
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