Low light optical detection of whole-cell bioluminescent bacterial bioreporters for visualisation of surface decontamination using nonthermal plasma

Robinson, Gareth M.; Thorn, Robin; Hancock, C.; Reynolds, Darren M.

Abstract

Introduction:
Maintenance of a clean, uncontaminated healthcare environment is instrumental in the delivery of modern healthcare. Nonthermal plasma is a technology with potential applications for sterilisation and disinfection in biological and medical fields. We describe the use of Salmonella enterica serotype Typhimurium DT104 modified with the Photorhabdus luminescens lux operon as a rapid in situ reporter of successful surface decontamination.

Method:
Plates of Salmonella Typhimurium DT104 pGLITE were treated a) immediately after inoculation or b) 24 hours post-inoculation with a HandiPlasma nonthermal plasma generator (Creo Medical, Chepstow, UK) and bioluminescence imaged in situ using an iXon EM+ DU-897 back-illuminated EMCCD camera (Andor, Belfast, UK). Alternatively, a 100 µL volume of liquid suspension of bacteria was deposited onto a 3D cast of a human hand, treated for 200 seconds and imaged immediately after.

Results and Discussion
Nonthermal plasma treatment of mature bacterial growth (high bacterial load) on agar plates, resulted in reduction in optical detection of light output compared to argon treated plates. Against freshly inoculated plates (moderate bacterial load), prior to incubation, non-thermal plasma prevented bacterial growth on the agar surface in a 25 mm diameter zone of inhibition around the applicator. This was not observed for the argon control (Fig 1). Against liquid culture inoculated onto a model polyurethane hand, non-thermal plasma produced a clear reduction in light output at the point of application, which was not observed for the argon control. The in situ optical detection of whole cell bioluminescent bacterial reporters has proven to be a flexible method for visualising decontamination on three dimensional surfaces, allowing real-time analysis of bacterial killing, in addition to spatial data regarding the location of contaminating bacteria.

• Presentation Conference Type: Conference Paper (unpublished)
• Conference Name: 3rd International Conference on Bio-Sensing Technology
• Start Date: May 12, 2013
• End Date: May 15, 2013
• Publication Date: May 12, 2013
• Peer Reviewed: Peer Reviewed
• Keywords: low light optical detection, whole-cell, bioluminescent bacterial bioreporters, visualisation, decontamination, nonthermal plasma
• Public URL: https://uwe-repository.worktribe.com/output/931930
• Related Public URLs: http://www.nature.com/natureevents/science/events/16200-3rd_International_Conference_on_Bio_Sensing_Technology
• Additional Information: Title of Conference or Conference Proceedings : 3rd International Conference on Bio-Sensing Technology