Viability of 3 h grown bacterial micro-colonies after direct Raman identification

• Only a small number of bacteria in the micro-colony are probed when using Raman micro-spectroscopy.
• Spectra containing discriminant information can be acquired at 3 h of growth on gelose.
• A small number of bacteria is affected or killed during laser exposure.
• Non-exposed bacteria are still alive and keep on growing.
• Growth delay is detectable for S. epidermidis but not for E. coli.

Clinical diagnostics in routine microbiology still mostly relies on bacterial growth, a time-consuming process that prevents test results to be used directly as key decision-making elements for therapeutic decisions. There is some evidence that Raman micro-spectroscopy provides clinically relevant information from a limited amount of bacterial cells, thus holding the promise of reduced growth times and accelerated result delivery. Indeed, bacterial identification at the species level directly from micro-colonies at an early time of growth (6 h) directly on their growth medium has been demonstrated. However, such analysis is suspected to be partly destructive and could prevent the further growth of the colony needed for other tests, e.g. antibiotic susceptibility testing (AST). In the present study, we evaluated the effect of the powerful laser excitation used for Raman identification on micro-colonies probed after very short growth times. We show here, using envelope integrity markers (Syto 9 and Propidium Iodide) directly on ultra-small micro-colonies of a few tens of Escherichia coli and Staphylococcus epidermidis cells (3 h growth time), that only the cells that are directly impacted by the laser lose their membrane integrity. Growth kinetics experiments show that the non-probed surrounding cells are sometimes also affected but that the micro-colonies keep their ability to grow, resulting in normal aspect and size of colonies after 15 h of growth. Thus, Raman spectroscopy could be used for very early (< 3 h) identification of grown micro-organisms without impairing further antibiotics susceptibility characterization steps