doi:10.1016/j.femsle.2004.11.024 
Copyright © 2004 Federation of European Microbiological Societies Published by Elsevier B.V.
The presence of the tet gene from cloning vectors impairs Salmonella survival in macrophages
Stephanie Abromaitisa, b, Sébastien Faucherc, Maxime Bélandc, Roy Curtiss IIIa and France Daiglea, c, 
, 
aWashington University, USA
bUniversity of California-Berkeley, USA
cUniversité de Montréal, Département de microbiologie et immunologie, CP 6128, succ. Centre-ville, Montréal, Qué., Canada H3C 3J7
Received 2 November 2004;
accepted 9 November 2004.
Available online 24 November 2004.
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Abstract
Cloning, mutagenesis and complementation of virulence factors are key steps to understand the mechanisms of bacterial pathogenesis and cloning vectors are routinely utilized for these processes. We have investigated the effect of the presence of commonly used cloning vectors on the survival of the intracellular bacterial pathogen Salmonella during macrophage infection. We demonstrate that the presence of the pSC101 derived tetracycline resistance gene on plasmids causes a lower survival rate of Salmonella in macrophages. The decrease in survival caused by the presence of the tet gene was not due to a higher susceptibility to gentamicin, a growth defect, or to increased sensitivity to acid. Higher susceptibility to hydrogen peroxide was observed in vitro for strain containing plasmid with the tet gene when the strains were grown at high densities but not when they were grown at low densities. Our findings demonstrate that the use of the tet gene for mutation or complementation can have deleterious effects and should thus be carefully considered.
Keywords: Plasmid effect; Tetracycline; Salmonella; Survival; Pathogenesis
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Bacterial strains, plasmids and growth conditions
- 2.2. Cell culture and macrophage infection
- 2.3. Molecular biology methods
- 2.4. Gentamicin sensitivity assay
- 2.5. Acid tolerance response
- 2.6. Hydrogen peroxide sensitivity assay
- 3. Results
- 3.1. Effect of commonly used plasmid vectors
- 3.2. Effect of the tetracycline gene during macrophage infection
- 3.3. Growth profile
- 3.4. In vitro stresses
- 4. Discussion
- Acknowledgements
- References
Fig. 1. Survival assays. Comparison of the abilities of the wild-type ISP1820 with or without plasmid to survive within THP-1 macrophage-like cells. For each strain at each time point, the number of bacteria recovered was expressed as a percentage of the number that were present in the inoculum. The values for percent recovery were normalized relative to that of the wild-type control (plasmid-free), which was designated 100% at each time point. The results represent an average of at least three distinct trials done in duplicate. Standard error bars are indicated. Strains carrying plasmid that demonstrated significant (p < 0.05) difference to the plasmid-free strain are indicated (*).
Fig. 2. Role of the tet gene during macrophage infection. Comparison of the abilities of bacteria carrying plasmid with or without the tet gene to survive within THP-1 macrophage-like cells. The results represent an average of at least three distinct trials done in duplicate. Standard error bars are indicated. Bacteria carrying plasmid that demonstrated a significant difference (p < 0.05) to the plasmid-free strain are indicated (*).
Fig. 3. Growth profile of strains carrying plasmid with or without the tet gene cultured in LB broth.
Fig. 4. Antibiotic susceptibility. Minimal inhibitory concentration (MIC) of (A) gentamicin or (B) polymyxin for strain carrying plasmid with or without the tet gene. The MIC for gentamicin of the strains carrying plasmid with the tet gene is significantly lower than the MIC of the strain carrying plasmid without the tet gene (p < 0.05).
Fig. 5. Survival assays using polymyxin. Comparison of the abilities of wild-type strain ISP1820 with or without plasmid to survive within THP-1 macrophage-like cells. See Fig. 1 legend. Strains carrying plasmid that demonstrated significant (p < 0.05) difference to the plasmid-free strain are indicated (*).
Fig. 6. Hydrogen peroxide sensitivity assay for strains cultured overnight carrying plasmids with or without the tet gene in presence of 400 μM of H2O2.
Table 1.
Bacterial strains and plasmids
Abstract
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