Early detection of Coxiella burnetii growth on axenic media using scanning electron microscopy

Coxiella burnetii, the causative agent of Q fever, is a highly infectious obligate intracellular bacterium. In consequence, cultivating this pathogen was fastidious and resource-consuming, requiring 3 weeks for isolation in cell culture. In recent years, axenic media allowing C. burnetii growth without eukaryotic cells were developed. These media reduced the complexity of its manipulation. However, despite this advancement, traditional growth monitoring methods such as cell culture, colony counts, and immunofluorescence remain time-consuming, requiring an average of 10-14 days to detect growth. However, this is still considered too long in the context of research, epidemiologic surveillance, and treatment failure. Here, we present a novel method that coupled axenic growth to early detection using the TM4000Plus II-Tabletop scanning electron microscopy (SEM) (Hitachi, Japan). For this, we used eight strains of C. burnetii that were cultured in axenic medium and then prepared for SEM observation. Growth was assessed by the calculation of the percentage area coverage. We were able to detect growth within only 3 days with a percentage area coverage superior to 60%. This result was reproducible among the eight strains. Finally, the use of SEM for early growth detection of C. burnetii in axenic medium is simple and feasible and may reduce detection time by 70%. This promising approach may also provide deeper insights into the morphological details, metabolic, and growth kinetics of this intracellular bacterium.IMPORTANCECoxiella burnetii is the causative agent of Q fever, a serious disease affecting humans and other mammals. Q fever outbreaks are reported worldwide, posing significant public health and economic concerns. While cultivating this pathogen is not typically required for diagnosis, it remains valuable in cases involving new clinical presentation, treatment failure, or atypical epidemiological situations. This highlights the need for novel techniques that are both simple and capable of enabling early growth detection. Here, we present an innovative and simple approach allowing early growth detection of C. burnetii in axenic media using scanning electron microscopy.