Campylobacter jejuni biofilm assessment by Nanoluc luciferase assay

Čukajne, Tjaša (author), Štravs, Petra (author), Sahin, Orhan (author), Zhang, Qijing (author), Berlec, Aleš (author), Klančnik, Anja (author)

Campylobacter jejuni, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into C. jejuni biofilm dynamics that could indirectly help in the development of improved food safety measures.

• Language: English
• Keywords: bioluminescence, bioflm assay, NanoLuc, viable but nonculturable, food safety
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 10 str.
• Numbering: Vol. 15, iss. 4, art. e5192
• PID: 20.500.12556/RUL-175367
• UDC: 579.2.088
• ISSN on article: 2331-8325
• DOI: 10.21769/BioProtoc.5192
• COBISS.SI-ID: 221752067
• Publication date in RUL: 24.10.2025

Record is a part of a journal

• Title: Bio-protocol
• Shortened title: Bio-protoc.
• Publisher: Bio-protocol LLC
• ISSN: 2331-8325
• COBISS.SI-ID: 8733561

Licences

• License: CC BY-NC 4.0, Creative Commons Attribution-NonCommercial 4.0 International
• Link: http://creativecommons.org/licenses/by-nc/4.0/
• Description: A creative commons license that bans commercial use, but the users don’t have to license their derivative works on the same terms.

Secondary language

• Language: Slovenian
• Keywords: Campylobacter jejuni, bioluminiscenca, biofilm, luciferaza NanoLuc, varnost živil