Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

Xu, Luping; Bai, Xingjian; Tenguria, Shivendra; Yi, Liu; Drolia, Rishi; Bhunia, Arun K.

doi:10.3389/fmicb.2020.575615

Cited by 8 publications

(6 citation statements)

References 56 publications

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“…Other methods such as fiber optic biosensors [8][9][10] and optical scattering technology [39,40] can detect pathogens, and some approaches can screen for phenotypes such as whether an antibiotic-induced stress response occurred in bacteria [41]. In contrast, cell-based methods are highly attractive tools for samples that may contain unknown and emerging human pathogens [42]. Unlike molecular methods that may detect dead bacterial organisms [43,44], cell-based assays are functional screens.…”

Section: Discussionmentioning

confidence: 99%

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Phillips¹,

Bergstrom²,

Walker³

et al. 2022

Pathogens

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Cell culture systems have greatly expanded our understanding of how bacterial pathogens target signaling pathways to manipulate the host and cause infection. Advances in genetic engineering have allowed for the creation of fluorescent protein readouts within signaling pathways, but these techniques have been underutilized in pathogen biology. Here, we genetically engineered a lung cell line with fluorescent reporters for extracellular signal-related kinase (ERK) and the downstream transcription factor FOS-related antigen 1 (Fra1) and evaluated signaling after inoculation with pathogenic and non-pathogenic bacteria. Cells were inoculated with 100 colony-forming units of Acinetobacter baylyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus agalactiae, or Staphylococcus epidermidis and imaged in a multi-mode reader. The alamarBlue cell viability assay was used as a reference test and showed that pathogenic P. aeruginosa induced significant (p < 0.05) cell death after 8 h in both wild-type and engineered cell lines compared to non-pathogenic S. epidermidis. In engineered cells, we found that Fra1 signaling was disrupted in as little as 4 h after inoculation with bacterial pathogens compared to delayed disruption in signaling by non-pathogenic S. epidermidis. Overall, we demonstrate that low levels of pathogenic versus non-pathogenic bacteria can be rapidly and sensitively screened based on ERK-Fra1 signaling.

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Section: Discussionmentioning

confidence: 99%

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Phillips¹,

Bergstrom²,

Walker³

et al. 2022

Pathogens

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“…Similar to other biosensors, living cells as CBB's receptors are isolated and immobilized on the surface of its accompanying transducer (Liu and Wang, 2009). Xu et al (2020) utilized a formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line (HCT-8) to capture viable pathogens in combination with antibodies. S. Enteriditis was used to inoculate various food samples artificially.…”

Section: Cell-based Biosensorsmentioning

confidence: 99%

Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens

et al. 2022

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Traditional foodborne pathogen detection methods are highly dependent on pre-treatment of samples and selective microbiological plating to reliably screen target microorganisms. Inherent limitations of conventional methods include longer turnaround time and high costs, use of bulky equipment, and the need for trained staff in centralized laboratory settings. Researchers have developed stable, reliable, sensitive, and selective, rapid foodborne pathogens detection assays to work around these limitations. Recent advances in rapid diagnostic technologies have shifted to on-site testing, which offers flexibility and ease-of-use, a significant improvement from traditional methods’ rigid and cumbersome steps. This comprehensive review aims to thoroughly discuss the recent advances, applications, and limitations of portable and rapid biosensors for routinely encountered foodborne pathogens. It discusses the major differences between biosensing systems based on the molecular interactions of target analytes and biorecognition agents. Though detection limits and costs still need further improvement, reviewed technologies have high potential to assist the food industry in the on-site detection of biological hazards such as foodborne pathogens and toxins to maintain safe and healthy foods. Finally, this review offers targeted recommendations for future development and commercialization of diagnostic technologies specifically for emerging and re-emerging foodborne pathogens.

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“…Methods have been developed to detect viable bacterial cells using fluorescent dyes such as SYTO 9 and propidium iodide ( Ou et al, 2019 ), and mammalian cell-based immunoassay ( Xu et al, 2020 ). Although they can detect or differentiate live cells from dead cells, these methods require many steps and special equipment.…”

Section: Introductionmentioning

confidence: 99%

A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder

Charlermroj

Makornwattana²,

Phuengwas³

et al. 2022

Front. Microbiol.

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Specific antibodies are essential components of immunoassay, which can be applied for the detection of pathogens. However, producing an antibody specific to live bacterial pathogens by the classical method of immunizing animals with live pathogens can be impractical. Phage display technology is an effective alternative method to obtain antibodies with the desired specificity against selected antigenic molecules. In this study, we demonstrated the power of a microarray-based technique for obtaining specific phage-derived antibody fragments against Salmonella, an important foodborne pathogen. The selected phage-displayed antibody fragments were subsequently employed to develop a lateral flow test strip assay for the detection of live Salmonella. The test strips showed specificity to Salmonella Enteritidis without cross-reactivity to eight serovars of Salmonella or other bacteria strains. The test strip assay requires 15 min, whereas the conventional biochemical and serological confirmation test requires at least 24 h. The microarray screening technique for specific phage-based binders and the test strip method can be further applied to other foodborne pathogens.

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Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

Cited by 8 publications

References 56 publications

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Engineered Cell Line Imaging Assay Differentiates Pathogenic from Non-Pathogenic Bacteria

Advances, applications, and limitations of portable and rapid detection technologies for routinely encountered foodborne pathogens

A rapid colorimetric lateral flow test strip for detection of live Salmonella Enteritidis using whole phage as a specific binder

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