Stably Luminescent Staphylococcus aureus Clinical Strains for Use in Bioluminescent Imaging

Abstract

In vivo bioluminescent imaging permits the visualization of bacteria in live animals, allowing researchers to monitor, both temporally and spatially, the progression of infection in each animal. We sought to engineer stably luminescent clinical strains of Staphylococcus aureus, with the goal of using such strains in mouse models. The gram-positive shuttle vector pMAD was used as the backbone for an integration plasmid. A chloramphenicol resistance gene, a modified lux operon from Photorhabdus luminescens, and approximately 650 bp of homology to the chromosome of the USA300 S. aureus strain NRS384 were added, generating plasmid pRP1195. Electroporation into strain RN4220 followed by temperature shift led to integration of pRP1195 into the chromosome. The integrated plasmid was transferred to clinical strains by phage transduction. Luminescent strains displayed no in vitro growth defects. Moreover, luminescence was stable in vitro after three rounds of subculture over 48 hours of growth in the absence of antibiotics. Mice were infected with a luminescent strain of NRS384 in skin and intravenous models. In a mouse skin model, luminescent bacteria were present in lesions that formed and cleared over the course of several days, and in an intravenous model, bacteria inoculated in the mouse tail vein were observed spreading to multiple tissues. No statistically significant difference in virulence was observed between NRS384 and the luminescent strain in either infection model. These preliminary data suggest that this luminescent USA300 strain is suitable for use in mouse models. Similar strains were engineered using other sequenced clinical strains. Because these strains are stably luminescent, they should prove useful in animal models of infection.

Mouse model of hematogenous implant-related Staphylococcus aureus biofilm infection reveals therapeutic targets

“…Here we report the development of a mouse model of a hematogenous orthopedic implant infection using a virulent bioluminescent CA-MRSA strain (43) and in vivo whole-animal optical imaging to monitor the dynamics of the infection beginning with bacteremia and resulting in seeding and infection of the surgically placed implant and surrounding bone and joint tissue. This model provided several insights into factors that contribute to the pathogenesis of hematogenous orthopedic infections.…”

“…aureus Strain. The bioluminescent SAP231 strain was used in all in vivo experiments and was generated as previously described from NRS384, a welldescribed USA300 CA-MRSA isolate obtained from a skin infection outbreak in the Mississippi prison system (43). SAP231 possesses a stably integrated modified luxABCDE operon from the bacterial insect pathogen Photorhabdus luminescens.…”

Staphylococcus aureus Epicutaneous Exposure Drives Skin Inflammation via IL-36-Mediated T Cell Responses

“…Both strains were derived from the parent S. aureus strain USA300 LAC, a well-described community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate that was obtained from an S. aureus skin and soft tissue infection outbreak in the Los Angeles County (LAC) Jail. Both LAC4303 and USA300 LAC:: lux possess a modified luxABCDE operon from the bacterial insect pathogen, Photorhabdus luminescens (Plaut et al, 2013; Thurlow et al, 2011). USA300 LAC:: lux was previously generated (Thurlow et al, 2011).…”

“…USA300 LAC:: lux was previously generated (Thurlow et al, 2011). LAC4303 was generated from S. aureus strain JE2 (BEI Resources), which is the LAC strain but cured of its native plasmids (Fey et al, 2013), by integrating plasmid pRP1195 into the chromosome a previously described (Plaut et al, 2013). S. aureus PSMα and δ-toxin mutant strains (engineered from LAC) were kindly provided by Dr. Michael Otto (NIH/NIAID).…”

A stable luciferase reporter plasmid for in vivo imaging in murine models of Staphylococcus aureus infections

“…This approach allows monitoring the progression of the infection in mice (Gahan 2012;Kuklin et al 2003;Melican and Richter-Dahlfors 2009;Plaut et al 2013) or the spatial and temporal Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-7229-2) contains supplementary material, which is available to authorized users. functionality of specific bacterial promoters (Lane et al 2007; Morin and Kaper 2009;Nesta et al 2014).…”