High-Content Imaging to Phenotype Antimicrobial Effects on Individual Bacteria at Scale

Abstract

High-content imaging (HCI) is a technique for screening multiple cells in high resolution to detect subtle morphological and phenotypic variation. The method has been commonly deployed on model eukaryotic cellular systems, often for screening new drugs and targets. HCI is not commonly utilized for studying bacterial populations but may be a powerful tool in understanding and combatting antimicrobial resistance. Consequently, we developed a high-throughput method for phenotyping bacteria under antimicrobial exposure at the scale of individual bacterial cells. Imaging conditions were optimized on an Opera Phenix confocal microscope (Perkin Elmer), and novel analysis pipelines were established for both Gram-negative bacilli and Gram-positive cocci. The potential of this approach was illustrated using isolates of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. HCI enabled the detection and assessment of subtle morphological characteristics, undetectable through conventional phenotypical methods, that could reproducibly distinguish between bacteria exposed to different classes of antimicrobials with distinct modes of action (MOAs). In addition, distinctive responses were observed between susceptible and resistant isolates. By phenotyping single bacterial cells, we observed intrapopulation differences, which may be critical in identifying persistence or emerging resistance during antimicrobial treatment. The work presented here outlines a comprehensive method for investigating morphological changes at scale in bacterial populations under specific perturbation. IMPORTANCE High-content imaging (HCI) is a microscopy technique that permits the screening of multiple cells simultaneously in high resolution to detect subtle morphological and phenotypic variation. The power of this methodology is that it can generate large data sets comprised of multiple parameters taken from individual cells subjected to a range of different conditions. We aimed to develop novel methods for using HCI to study bacterial cells exposed to a range of different antibiotic classes. Using an Opera Phenix confocal microscope (Perkin Elmer) and novel analysis pipelines, we created a method to study the morphological characteristics of Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus when exposed to antibacterial drugs with differing modes of action. By imaging individual bacterial cells at high resolution and scale, we observed intrapopulation differences associated with different antibiotics. The outlined methods are highly relevant for how we begin to better understand and combat antimicrobial resistance.

Inhibitory Concentrations of Ciprofloxacin Induce an Adaptive Response Promoting the Intracellular Survival of Salmonella enterica Serovar Typhimurium

“…Ciprofloxacin induces morphological changes in S. Typhimurium. To better understand the impact of ciprofloxacin on the selected organisms, we exposed organisms D23580, SL1344, and VNS20081 to 0Â, 1Â, 2Â, or 4Â MIC of ciprofloxacin for 2 h and then imaged them using a quantitative high-content confocal microscopy system (24,65). A time point of 2 h was selected to capture early adaptive responses.…”

“…Opera Phenix confocal microscopy image analysis of single bacteria. Images generated on the Opera Phenix were analyzed using the Harmony software (Perkin Elmer), as previously described (24,65). Briefly, inputted images underwent flatfield correction, and images were calculated using the DAPI and Alexa Fluor 647 channels and then refined by size and shape characteristics.…”

Rapid Detection of Imipenem Resistance in Gram-Negative Bacteria Using Tabletop Scanning Electron Microscopy: A Preliminary Evaluation

“…Moreover, other studies recently evaluated the phenotypic response of different bacterial species using phase contrast microscopy. Diverse abnormal morphologies were associated to K. pneumoniae after incubation with a panel of antibiotics, notably analyzing the length to width ratio and cell roundness among other parameters (Sridhar et al, 2021 ). Also, bulging was noticed in E. coli isolates after a short incubation with cefsulodin, another β-lactam antibiotic, and was observed right before the cell lysis (Zahir et al, 2019 ).…”

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

“…More recently, high-throughput imaging has made it possible to rapidly screen hundreds to thousands of samples. Still, high-throughput imaging has primarily been used to evaluate compounds with antibacterial potential in pathogen research [19,20]. These techniques could create high-throughput imaging assays to screen for pathogens if distinguishing cellular signals are known.…”