Abstract
BackgroundThe green fluorescence protein (GFP)-associated fluorescence method and the luciferase-associated bioluminescence method are the two major methods for IVIS imaging system to investigate the bacterial infection in animal models. The aim of this study was to evaluate the infection route of Gram-negative bacteria carrying a stable and broad range of conjugative bioluminescence plasmid pSE-Lux1 in a mouse model.ResultsBoth encapsulated and non-encapsulated Gram-negative bacteria were used as hosts to evaluate conjugation efficiency and plasmid stability of pSE-Lux1, a recombinant of pSE34 and luxABCDE operon. The plasmid conjugation efficiencies of pSE-Lux1 ranged from 10−3 to 10−7 in various Gram-negative bacteria. Plasmid pSE-Lux1 maintained in Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica serovars Choleraesues (abbreviated S. Choleraesuis) and Typhimurium (S. Typhimurium), than in Acinetobacter baumannii and Serratia marcescens, was shown to be of better stability for at least four days. To investigate systemic bacterial infections, K. pneumoniae strain CG354 was intravenously injected, and then was clearly observed to be non-pathogenic to Balb/c mice for a long-term bioluminescence monitoring for 6 days. For examining dynamic distributions of gastrointestinal tract infection, the invasion protein SipB-deficient mutant OU5045△sipB and OU5046△sipB of S. serovar Typhimurium constructed in this study, compared to wild-type strain OU5045 and its virulence plasmid-less strain OU5046, were of less virulence to mice.ConclusionsThis is the first study to evaluate the conjugative and stable bioluminescence vehicle system of pSE-Lux1 in a wide range of Gram-negative bacteria, a system that can provide a useful reporter approach to trace systemic and gastrointestinal bacterial infections in a mouse model.
Highlights
The green fluorescence protein (GFP)-associated fluorescence method and the luciferase-associated bioluminescence method are the two major methods for IVIS imaging system to investigate the bacterial infection in animal models
Many luxABCDE-carrying vectors have been constructed for bioluminescence assays; a steady, broad-host auto-bioluminescence vehicle is needed, especially a conjugatable plasmid is preferred to deliver exogenous DNA between bacteria and overcome the difficulty in transformation for some thick-capsule bacteria, such as Klebsiella pneumoniae [2,4]
The bacterial conjugation efficiency and plasmid stability of pSE-Lux1 were determined in various Gram-negative bacteria, and distributions of the pSE-Lux1-carrying bacteria in Balb/c mice were evaluated by an in vivo imaging system
Summary
The green fluorescence protein (GFP)-associated fluorescence method and the luciferase-associated bioluminescence method are the two major methods for IVIS imaging system to investigate the bacterial infection in animal models. The aim of this study was to evaluate the infection route of Gram-negative bacteria carrying a stable and broad range of conjugative bioluminescence plasmid pSE-Lux in a mouse model. We constructed a conjugative vehicle pSE-Lux, which is a chimeric vector in combination of a stable conjugative native pSE34 that include pilX1, pilX2, pilX4, pilX5, pilX6, pilX7, pilX8, pilX9, pilX10, pilX11, taxA, taxB, and tax of type IV secretion system, with a bioluminescence reporter p3ZLux that contains the luxABCDE-kan operon of pXen-5 (Bioware, Caliper Life Sciences, USA) and the portion of pGEM®-3Z (Promega Corporation, USA) with 300–400 copies per bacterial cell [5,6,7,8]. The bacterial conjugation efficiency and plasmid stability of pSE-Lux were determined in various Gram-negative bacteria, and distributions of the pSE-Lux1-carrying bacteria in Balb/c mice were evaluated by an in vivo imaging system
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