1 Department of Medical Microbiology, The University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, UK; 2 Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium
Received 8 October 2001; returned 24 January 2002; revised 25 March 2002; accepted 6 June 2002
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Abstract |
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Introduction |
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Recently, the taxonomy of the B. cepacia complex has undergone major revisions. Integrated genotypic and phenotypic analyses have shown that isolates identified previously as B. cepacia comprise a diverse group of at least eight genomic species, or genomovars.4 Several genomovars can be distinguished phenotypically and have been awarded species status; these include genomovar II (Burkholderia multivorans), genomovar IV (Burkholderia stabilis), genomovar V (Burkholderia vietnamiensis) and genomovar VII (Burkholderia ambifaria). These four species, together with the B. cepacia genomovars I, III, VI and VIII, presently constitute the B. cepacia complex.4
Interest in the B. cepacia complex originates not only from the groups role as human, animal and plant pathogens, but also from the antifungal and metabolic potential of these bacteria. For example, the complex includes strain AMMD (B. ambifaria), which is highly effective as a biopesticide used to control soil-borne plant diseases caused by fungi and oomycetes.5
Although all genomovars of the B. cepacia complex have been cultured from the sputa of CF patients,4 genomovar III and B. multivorans account for >90% of isolates recovered from patients in North America5,6 and Europe (unpublished data). These bacteria are also those most frequently associated with epidemic spread and with cepacia syndrome, a severe progressive respiratory failure with bacteraemia.4
Previous studies of the antimicrobial susceptibility of B. cepacia were performed before recent taxonomic revisions.2,3 The purpose of this study was to determine the in vitro susceptibility of representative isolates of the complex to major classes of antimicrobials, including agents used in the treatment of infections caused by Pseudomonas species.
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Materials and methods |
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The 65 B. cepacia complex strains used included the 30 isolates of the B. cepacia complex strain panel7 and 12 genomovar III isolates recently cultured from soils in France and Australia.8 Isolates were identified provisionally using the API 20 NE system (bioMérieux, Marcy lÉtoile, France) and confirmed by recA restriction fragment length polymorphisms or protein analysis and DNA:DNA hybridization.4 Clonal relationships were excluded by PFGE fingerprinting3 (PFGE, CHEF, Bio-Rad Laboratories). The exceptions were BC7, J2315 and K56-2, which represent the major transmissible lineage known as ET12.7 Pseudomonas aeruginosa NTCC 10662 was used as a reference strain; currently, there is no reference strain for the B. cepacia complex relating to in vitro antimicrobial susceptibility tests.
Antimicrobial agents
All compounds were obtained from Sigma (Poole, UK), with the exceptions of ceftazidime (GlaxoSmithKline, Uxbridge, UK) and ciprofloxacin (Bayer, Wuppertal, Germany), and were prepared following the manufacturers instructions. Stock solutions were filter-sterilized (pore size 0.2 µm; Millipore), stored at 70°C, and used within the recommended period.
Susceptibility testing
MICs were determined on IsoSensitest agar (Oxoid) using agar dilution. Final concentrations of each antimicrobial were 0.06128 mg/L. Plates were dried and stored overnight at 4°C before use. Bacteria were grown overnight at 37°C in nutrient broth supplemented with 0.5% yeast extract (NBYE, Oxoid) in an orbital incubator at 140 rpm. A final inoculum of 104 cfu/spot was delivered by multipoint inoculator (Denley, Billinghurst, UK) and the plates were incubated overnight at 37°C in air. MIC was defined as the lowest concentration of antibiotic to inhibit bacterial growth, and resistance as an MIC greater than the BSAC breakpoints for Pseudomonas species.9 Statistical analysis was performed using the 2 test, and a P value <0.05 was taken to indicate significance.
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Results |
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The MICs of ceftazidime and trimethoprim for clinical and soil isolates (identified in Table 1 by prefix R) of B. cepacia genomovar III did not differ significantly. However, environmental strains were more susceptible to ciprofloxacin and chloramphenicol than clinical isolates (P = 0.021 and 0.002, respectively). Clinical and environmental isolates of B. vietnamiensis were also more susceptible to ceftazidime (P = 0.04) and choramphenicol (P = 0.004) than strains from other genomovars.
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Discussion |
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Clinical isolates are typically thought to be more resistant to antibiotics than environmental isolates because of previous antibiotic exposure. Thus, the similarity in the susceptibility of clinical and environmental isolates of genomovar III to trimethoprim and ceftazidime is interesting. In the 1980s, trimethoprim was used as first-line choice against B. cepacia, and ceftazidime is currently considered to be one of the few antimicrobials effective against B. cepacia complex infections.
In risk assessment of biological control strains,5 it has been argued that candidate biopesticide strains should be susceptible to the antibiotics used in clinical practice, and have a low potential to develop resistance. Strains of B. vietnamiensis and B. ambifaria have been favoured as biopesticides and bioremediators because of their low incidence of isolation from CF patients.5 In our study, we found B. vietnamiensis strains to be more susceptible to ceftazidime and chloramphenicol than strains from other genomovars. Thus, it could be argued that B. vietnamiensis strains are safe candidates for large-scale commercial application. However, an important consideration is that such strains could acquire resistance by mutation or horizontal gene transfer.
We have previously reported in vitro transfer of ceftazidime resistance by transduction in the B. cepacia complex.10 Although acquired resistance was not a subject of the present study, we found evidence of mutation to ceftazidime resistance (six-fold or greater increase in MIC compared with parent strain) in the biopesticide strain AMMD, in strains of B. vietnamiensis and in the other genomovars included in this study.
Lack of concordance in the MICs of ciprofloxacin, tobramycin, tetracycline and trimethoprim shown for the ET12 representatives, BC7, J2315 and K56-2, was interesting. K56-2 (MIC 1 mg/L) was also more susceptible to meropenem than BC7 and J2315 (MIC 8 mg/L). These differences in susceptibility suggest that further investigations of these closely related and well-studied strains (whole-genome sequencing of J2315 is near completion, see http://www.sanger.ac.uk/Projects/) could reveal mechanisms of antibiotic susceptibility and resistance in this important genomovar III lineage.
To our knowledge, this is one of the first reports of antimicrobial susceptibility to take account of the new taxonomic groups within the B. cepacia complex. In this study, we focused on the B. cepacia complex strain panel, and on the two groups, B. multivorans and B. cepacia genomovar III, that are most associated with human infections. Interestingly, the results confirm our earlier observation of the resistance of the lineage (later to be known as ET12) to most antimicrobials, including meropenem.2 Unfortunately, the number of isolates belonging to B. stabilis and B. ambifaria available for study is limited. In addition, to our knowledge, environmental isolates of B. multivorans are scarce. Despite these limitations, our study provides a useful framework for further studies of the mechanisms of susceptibility and resistance in this highly adaptable and expanding group of organisms.
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Acknowledgements |
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Footnotes |
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References |
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3 . Pitt, T. L., Kaufmann, M. E., Patel, P. S., Benge, L. C. A., Gaskin, S. & Livermore, D. M. (1996). Type characterisation and antibiotic susceptibility of Burkholderia (Pseudomonas) cepacia isolates from patients with cystic fibrosis in the United Kingdom and the Republic of Ireland. Journal of Medical Microbiology 44, 20310.[Abstract]
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