High-Throughput Quantification of Bacterial-Cell Interactions Using Virtual Colony Counts

Abstract

The quantification of bacteria in cell culture infection models is of paramount importance for the characterization of host-pathogen interactions and pathogenicity factors involved. The standard to enumerate bacteria in these assays is plating of a dilution series on solid agar and counting of the resulting colony forming units (CFU). In contrast, the virtual colony count (VCC) method is a high-throughput compatible alternative with minimized manual input. Based on the recording of quantitative growth kinetics, VCC relates the time to reach a given absorbance threshold to the initial cell count using a series of calibration curves. Here, we adapted the VCC method using the model organism Salmonella enterica sv. Typhimurium (S. Typhimurium) in combination with established cell culture-based infection models. For HeLa infections, a direct side-by-side comparison showed a good correlation of VCC with CFU counting after plating. For MDCK cells and RAW macrophages we found that VCC reproduced the expected phenotypes of different S. Typhimurium mutants. Furthermore, we demonstrated the use of VCC to test the inhibition of Salmonella invasion by the probiotic E. coli strain Nissle 1917. Taken together, VCC provides a flexible, label-free, automation-compatible methodology to quantify bacteria in in vitro infection assays.

Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms

“…Such assays are laborious, exhibit limited precision, and are not easily scalable. Some larger-scale methods have been developed recently (33–35), but these are still subject to the inherent experimental noise that stems from well-to-well, plate-to-plate, or batch-to-batch variation.…”

Optimizing concentrations and contact times of cleaning and sanitizing agents for inactivating winery spoilage microorganisms

“…Fluorescent stain data is rapidly obtained, requiring only minutes for scans using a microplate reader. Fluorescent staining with propidium iodide is a well-established method, but the selective exclusion of propidium iodide depends on the loss of cellular membrane integrity and control [20]. Therefore, only a limited number of cleaning and sanitizing agents can be used that have modes of action primarily affecting membrane permeability.…”

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria

“…The main method used to quantify facultative intracellular bacteria is to lyze infected cell monolayers using a low concentrated detergent. The cell lysates were then serially diluted and spot-plated for colony forming units (CFUs) count [5, 11]. This conventional technique has serious drawbacks, such as the incomplete lysis of eukaryotic cells, the type of detergent used for cell lysis that could interfere with bacterial viability, the choice of appropriate dilution for counting, the unsuitability for high-throughput analyses, the variable incubation times and the coalescence of colonies, leading to unreliable and ambiguous results [5, 12, 13].…”

“…The cell lysates were then serially diluted and spot-plated for colony forming units (CFUs) count [5, 11]. This conventional technique has serious drawbacks, such as the incomplete lysis of eukaryotic cells, the type of detergent used for cell lysis that could interfere with bacterial viability, the choice of appropriate dilution for counting, the unsuitability for high-throughput analyses, the variable incubation times and the coalescence of colonies, leading to unreliable and ambiguous results [5, 12, 13]. Moreover, preparation of media, dilution tubes, as well as counting of colonies are time-consuming and labor-intensive [5, 12, 13].…”

“…Additionally, the CFU counting technique cannot be applied for obligate intracellular pathogens. Consequently, several alternative quantification methods, such as colorimetric and fluorescence-based methods have been developed [5, 14–21]. These techniques are mainly used for the quantification of obligate intracellular pathogens, such as Chlamydia trachomatis .…”