Automated, High-Throughput Detection of Bacterial Adherence to Host Cells

Summary

Detection of host-bacterial pathogen interactions based on phenotypic adherence using high-throughput fluorescence labeling imaging along with automated statistical analysis methods enables rapid evaluation of potential bacterial interactions with host cells.

Abstract

Identification of emerging bacterial pathogens is critical for human health and security. Bacterial adherence to host cells is an essential step in bacterial infections and constitutes a hallmark of potential threat. Therefore, examining the adherence of bacteria to host cells can be used as a component of bacterial threat assessment. A standard method for enumerating bacterial adherence to host cells is to co-incubate bacteria with host cells, harvest the adherent bacteria, plate the harvested cells on solid media, and then count the resultant colony forming units (CFU). Alternatively, bacterial adherence to host cells can be evaluated using immunofluorescence microscopy-based approaches. However, conventional strategies for implementing these approaches are time-consuming and inefficient. Here, a recently developed automated fluorescence microscopy-based imaging method is described. When combined with high-throughput image processing and statistical analysis, the method enables rapid quantification of bacteria that adhere to host cells. Two bacterial species, Gram-negative Pseudomonas aeruginosa and Gram-positive Listeria monocytogenes and corresponding negative controls, were tested to demonstrate the protocol. The results show that this approach rapidly and accurately enumerates adherent bacteria and significantly reduces experimental workloads and timelines.

Introduction

Bacterial adhesion is a process whereby bacteria attach to other cells or surfaces. Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in some cases, invasion of host cells^1,2,3. Emerging infectious diseases constitute major public health threats, as evidenced by the recent COVID-19 pandemic^4,5,6. Importantly, new or emerging pathogens may not be readily discerned using genomic-based approaches, especially in cas....

Protocol

1. A549 cell culture

1. Maintain the A549 cell line in F-12K medium supplemented with 10% fetal bovine serum (FBS) and incubate at 37 °C, 5% CO₂.
2. Change the medium every 3-4 days and passage at 85%-95% confluency.
3. Briefly, rinse the cells with 1 x phosphate-buffered saline (PBS, pH 7.4, unless otherwise indicated) and treat with 1 ml of 0.25% Trypsin-0.53 mM ethylenediaminetetraacetic acid (EDTA) solution (submerging the cell layer) for about 2 min at 37 °C.
4. Add additional 6 mL of complete growth medium (F-12K medium + 10% FBS) to stop the protease activity. Then, plate the cells in a sterile polystyrene T-75....

Results

To develop the fluorescence imaging-based bacterial adherence assay, P. aeruginosa strain PAO1 and its negative-adherence counterpart E. coli were used to test the protocol effectiveness, as the adherence of these bacteria to A549 cells had been reported^14,20,22. First, GFP- labeled P. aeruginosa (PAO1) and GFP-labeled E. coli were co-incubated with a human immortalized epithelial cell line A5.......

Discussion

The protocol describes an automated approach for enumerating bacterial attachment to host cells. The described approach has several attractive advantages over conventional methods. First, this approach enables the precise quantification of the number of microbial pathogen cells that are attached to individual host cells. Importantly, this quantification can be performed without the need for laborious bacterial harvesting, serial dilutions, plating on solid media, and determination of CFUs^10,<.......

Materials

Name	Company	Catalog Number	Comments
10x PBS	VWR	45001-130
4′,6-diamidino-2-phenylindole (DAPI)	Thermo Fisher	62248	Host cell staining dye
96 well plate	Corning	3882	Half area well, flat clear bottom
A549 cells	ATCC	CCL 185	Mammalian cell line
BactoView Live Red	Biotium	40101	Bacteria staning dye
Centrifuge	Eppendorf	5810R
CFSE cell division tracker	BioLegend	423801
Cytation 5	BioTek	Cytation 5	Cell imaging multi-mode reader
E. coli			Laboratory stock
EGM bulletKit	Lonza	CC-3124	HUVEC cell culture medium
EHEC			NIST collections
F-12k medium	ATCC	302004	A549 cell culture medium
Fetal bovine serum	Corning	35-016-CV
HUVEC			Laboratory stock
L. monocytogenes			NIST collections
OD600 DiluPhotometer	IMPLEN
P. aeruginosa			Dr. Lori Burrows laboratory stock
P. aeruginosa ΔpilA			Dr. Lori Burrows laboratory stock
S. agalactiae			NIST collections
S. aureus	BEI	NR-46543
S. aureus ΔsaeR	BEI	NR-48164
S. rubidaea			NIST collections
Typical soy broth	Growcells	MBPE-4040