Rapid label-free identification of seven bacterial species using microfluidics, single-cell time-lapse phase-contrast microscopy, and deep learning-based image and video classification.

For effective treatment of bacterial infections, it is essential to identify the species causing the infection as early as possible. Current methods typically require hours of overnight culturing of a bacterial sample and a larger quantity of cells to function effectively. This study uses one-hour phase-contrast time-lapses of single-cell bacterial growth collected from microfluidic chip traps, also known as a "mother machine". These time-lapses are then used to train deep artificial neural networks (Convolutional Neural Networks and Vision Transformers) to identify the species. We have previously demonstrated this approach on four different species, which is now extended to seven common pathogens causing human infections: Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, Proteus mirabilis, and Staphylococcus aureus. Furthermore, we expand upon our previous work by evaluating real-time performance as additional frames are captured during testing, and investigating the role of training set size, data quality, and data augmentation as well as the contribution of texture and morphology to performance. The experiments suggest that spatiotemporal features can be learned from video data of bacterial cell divisions, with both texture and morphology contributing to classifier decision. The method could be used simultaneously with phenotypic antibiotic susceptibility testing (AST) in the microfluidic chip. The best models attained an average precision of 93.5% and a recall of 94.7% (0.997 AUC) on a trap basis in a separate, unseen experiment with mixed species after around one hour. However, in a real-world scenario, one can assume many traps will contain the actual species causing the infection. Still, several challenges remain, such as isolating bacteria directly from blood and validating the method on diverse clinical isolates. This proof of principle study brings us closer to real-time diagnostics that could transform the initial treatment of acute infections.