1 Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049-Madrid; 2 Instituto de Investigaciones Biomédicas, CSIC/UAM, C/ Arturo Duperier 4, 28029-Madrid, Spain
Received 13 October 2003; returned 29 October 2003; revised 13 November 2003; accepted 17 November 2003
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Abstract |
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Results: Two S. maltophilia isogenic strains were analysed: the wild-type strain D457 and strain D457R, which is a SmeDEF overproducer. In co-culture experiments, under non-selective pressure the wild-type strain displaced the mutant strain D457R. Metabolic profiling showed that SmeDEF overproduction leads to several changes in S. maltophilia metabolism. Using a Dictyostelium discoideum model of bacterial virulence, we found overexpression of SmeDEF to be associated with a reduction in S. maltophilia virulence.
Conclusions: Together, these data indicate that overexpression of the multidrug efflux pump SmeDEF causes a metabolic burden for S. maltophilia.
Keywords: antibiotic resistance, S. maltophilia, MDR, bacterial fitness, metabolic profiling, virulence
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Introduction |
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Antibiotic resistance is very important for bacterial growth during antibiotic treatment. Nevertheless, it can cause a metabolic burden that can become onerous once the selective pressure is removed. Under such conditions, antibiotic-resistant bacteria can be displaced by their susceptible counterparts.
In earlier work, we characterized the SmeDEF efflux pump of S. maltophilia.6 This is involved both in intrinsic7 and acquired8 antibiotic resistance in this species. The present study was undertaken to establish the effect of the overexpression of SmeDEF on S. maltophilia fitness.
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Materials and methods |
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The strains used were S. maltophilia D457 and its multiresistant derivative, D457R. The latter overexpresses the smeDEF operon6 because it has a point mutation in smeT, the gene coding for the repressor of smeDEF expression.9 Both strains were grown in LB medium at 37°C unless otherwise indicated. Strains P. aeruginosa PAO1 and Klebsiella aerogenes were grown as described in the Dictyostelium discoideum assays below.
Growth competition between S. maltophilia D457 and S. maltophilia D457R
Growth competition experiments between D457 and D457R were performed in LB broth by mixing 106/mL cells of each strain and incubating them at 37°C with agitation. Every 24 h the bacterial mixture was diluted 1/1000 with pre-warmed LB broth. The representation of the strains in the culture was determined at different times by plating serial dilutions on both non-selective and selective LB agar plates containing 15 mg/L of nalidixic acid (nalidixic acid MIC: D457 = 8 mg/L, D457R = 128 mg/L).3 The plates with nalidixic acid only allowed the growth of D457R colonies. Both D457 and D457R colonies grew on the non-selective LB agar plates. As controls, the same experimental procedure was undertaken but using pure cultures of D457 and D457R. The number of nalidixic acid-resistant D457 mutants and the number of D457R cells that lost the MDR phenotype was <107 at all times.
Metabolic profiling
Overnight cultures of D457 and D457R were washed twice with 0.85% NaCl and diluted to OD550 0.1 (79%T) in 0.85% NaCl supplemented with trace elements. Bacterial suspension (150 µL) was poured into each well of a 96-well GN2 plate (Biolog, Hayward, CA, USA) and incubated at 37°C for 24 h. The GN2 plates have different carbon sources in each well and a metabolism reporter based on the reduction of tetrazolium coupled to bacterial respiration.10 The response of the cells was monitored colorimetrically using a Spectramax 250 (Molecular Devices Corporation, Sunnyvale, CA, USA) at 590 nm. This experiment was performed in triplicate.
Effect of S. maltophilia on D. discoideum growth
D. discoideum cells (strain AX4) were grown in HL5 medium at 22°C. The bacterial strains were grown at 37°C in SM (1% glucose, 1% peptone, 0.1% yeast extract, 0.1% MgSO4·6H2O, 0.19 % KH2PO4, 0.06 % K2HPO4, pH 6.5) to an optical density of 0.6 at 600 nm before plating.11
For quantitative virulence assays, the two S. maltophilia test strains, P. aeruginosa and K. aerogenes, were plated on SM agar and then spotted with serial dilutions of D. discoideum cells (4000 cells in drop 1, 2000 cells in drop 2, 1000 cells in drop 3, followed by a consecutive 2x dilution until drop 11 with two cells). The bacterial lawns were formed with a mixture containing K. aerogenes as the food source for D. discoideum plus the strain to be tested. All plates were incubated at 22°C for 5 days.11
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Results and discussion |
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To analyse whether overexpression of SmeDEF affects the fitness of S. maltophilia, we studied growth competition, under conditions of feast/famine13 that bacteria probably encounter in their natural environment. Figure 1 shows that within 2 weeks the wild-type strain progressively displaced the MDR strain to almost undetectable levels, indicating that overexpression of the MDR pump SmeDEF impairs the fitness of S. maltophilia.
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Conclusions |
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Acknowledgements |
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Footnotes |
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References |
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