Development of a Staphylococcus aureus reporter strain with click beetle red luciferase for enhanced in vivo imaging of experimental bacteremia\xa0and mixed infections

Robert J Miller,William R Anderson,Martin P Alphonse,Nicholas M Bernthal,Roger V Ortines,Lea Fortuno-Miranda,Jeffrey Zhang,Katrin Schilcher,Nathan K Archer,Momina Mazhar,Kevin P Francis,Dustin Dikeman ,Jeff W.m Bulte ,Isabelle Brown ,Bret L Pinsker,Daniel Joyce ,Alexander R Horswill,Yu Wang,Haiyun Liu,Julie Czupryna,Heidi A Crosby,Lloyd S Miller

doi:10.1038/s41598-019-52982-0

Abstract

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. However, the relatively short emission wavelength of lux (peak 490 nm) has limited tissue penetration. To overcome this limitation, the gene for the click beetle (Pyrophorus plagiophtalamus) red luciferase (luc) (with a longer >600 emission wavelength), was introduced singly and in combination with the lux operon into a methicillin-resistant S. aureus strain. After administration of the substrate D-luciferin, the luc bioluminescent signal was substantially greater than the lux signal in vitro. The luc signal had enhanced tissue penetration and improved anatomical co-registration with infected internal organs compared with the lux signal in a mouse model of S. aureus bacteremia with a sensitivity of approximately 3 × 104 CFU from the kidneys. Finally, in an in vivo mixed bacterial wound infection mouse model, S. aureus luc signals could be spectrally unmixed from Pseudomonas aeruginosa lux signals to noninvasively monitor the bacterial burden of both strains. Therefore, the S. aureus luc reporter may provide a technological advance for monitoring invasive organ dissemination during S. aureus bacteremia and for studying bacterial dynamics during mixed infections.

Highlights

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens
If the lux operon construct is stably integrated into the bacterial chromosome or into a stable plasmid, light production is maintained in all progeny and the bioluminescence imaging (BLI) signals highly correlate with ex vivo colony forming units (CFU)[13,17,18,19,20,21]
S. aureus bacteremia infections are a major clinical problem, especially since the mortality has remained extremely high and the treatment has been complicated by virulent and multi-drug resistant MRSA strains[3,4,5,6]

Summary

Introduction

In vivo bioluminescence imaging has been used to monitor Staphylococcus aureus infections in preclinical models by employing bacterial reporter strains possessing a modified lux operon from Photorhabdus luminescens. The use of in vivo BLI with bioluminescent S. aureus strains has permitted the noninvasive and longitudinal monitoring of the bacterial burden, which has provided key information about the infectious course and disease pathogenesis in skin and soft tissue infections[13,17,22,23,24,25,26] as well as musculoskeletal infections[16,19,27,28,29,30,31,32,33,34] This technology has been used to evaluate therapeutics, such as antibiotics[18,20,35,36,37,38,39], active and passive vaccines[29,40,41] and other antimicrobials[37,42] as well as S. aureus-specific diagnostic imaging probes[27,43,44]. The bioluminescent signals from these lux and luc expressing S. aureus strains were evaluated in vitro and in invasive (i.e., bacteremia) and more superficial (i.e., skin and musculoskeletal) preclinical models of S. aureus infection in vivo

Methods

Results

Conclusion