Cave microbial communities are structured by environmental matrix and depth and can be characterized with...

Terrestrial caves are unique ecosystems characterized by environmental stability, nutrient limitation, and absence of light, yet they host diverse microbial communities with ecological and public health relevance. Microbiome studies of caves have typically been limited in scope, sampling only select environments or cave depths. We conducted a broad survey of microbial diversity in three natural limestone caves and two abandoned mines spanning continental, subtropical, and arid climates in the United States. Using amplicon sequencing of 382 samples composed of soil, rock, water, air, and bat and rodent feces, microbiome composition was found to be primarily structured by cave location, followed by environmental matrix (soil, water, air, etc.), and transect distance from the cave entrance. Significant heterogeneity was observed both between and within caves, underscoring the need for spatially explicit and multi-matrix sampling to capture representative community profiles. Portable DNA extraction and quantitative PCR (qPCR) technologies for onsite detection of microbial pathogens were further validated, demonstrating comparable results to laboratory-based workflows and reducing sample-to-result turnaround time from several days to less than 2 h. Pathogen panels detected zoonotic and waterborne agents of human health concern, including Salmonella and Legionella, directly from cave environments. Collectively, our findings establish a methodological framework for robust microbiome characterization in subterranean ecosystems and highlight the feasibility of field-deployable genetic tools for both biodiversity mapping and rapid pathogen surveillance. These approaches will enable more systematic monitoring of cave environments, with applications for ecology, conservation, and public health.

Caves and mines represent extreme and isolated environments that harbor unique microbial communities, yet they remain among the least studied environments on Earth. Understanding how these communities are structured across different habitats and locations is essential for both ecological research and public health monitoring. In this study, we surveyed microbiomes across multiple caves and environmental materials to reveal how location, substrate type, and depth shape microbial diversity. We also demonstrated that portable DNA extraction and analysis tools can be used in the field to rapidly detect microorganisms, including potential pathogens, without the need for laboratory infrastructure. These results provide new insight into how microbial life is distributed in subterranean ecosystems and establish practical methods for monitoring microbial diversity and detecting pathogens in remote environments.