# DMEM and EMEM as alternate growth media for pathogenic Leptospira

## TL;DR

Researchers found that DMEM and EMEM media better mimic the host environment for pathogenic Leptospira, improving in vitro studies of leptospirosis.

## Contribution

Identified DMEM and EMEM as superior growth media for Leptospira that better reflect host conditions compared to traditional media.

## Key findings

- Leptospires cultured in DMEM and EMEM shared 40-47% of differentially expressed genes with those in host blood, compared to 20% in HAN.
- DMEM and EMEM cultures showed gene and pathway expression patterns closely matching those in host environments, including lipid A profiles.
- Using DMEM and EMEM at 37°C with 5% CO2 provides a cost-effective and reproducible method for leptospiral research.

## Abstract

Pathogenic Leptospira species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured in a laboratory setting under in vitro conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of in vitro studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available in vitro tools to evaluate leptospiral pathogenesis. We quantified genome-wide transcription of pathogenic Leptospira interrogans cultured in different in vitro media compositions and conditions—EMJH at 29 °C and DMEM, EMEM, and HAN at 37 °C and 5% CO2. Using EMJH as standard, we compared gene expression in these compositions to genome-wide transcription gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR < 0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for in vivo pathogenic studies than EMJH or HAN media in Leptospira. These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.

Leptospirosis is a life-threatening disease that can occur in diverse epidemiological settings and is the leading zoonotic disease in terms of morbidity and mortality worldwide. However, the major burden and impact of this neglected disease is in the world’s poorest countries. The burden of leptospirosis is likely to increase, as extreme weather events intensify in frequency with global climate change. The complexity of the genus and the tools to culture and isolate leptospires, especially on developing countries, thwarted research and the development of sensitive diagnostic assays and effective prevention methods. We evaluated different media and culture conditions to grow leptospires in vitro, including spirochetes harvest directly from the blood of infected hamsters, and compared to the traditional in vitro culturing of leptospires in EMJH medium at 30 °C. Using transcriptomic analysis and lipid A profiling, our results indicated that the growth of leptospires at 37 °C with 5% CO2 in EMEM and DMEM, traditional mammalian cell culture media, was not only feasible but also induced an overall gene expression and a lipid A profile similar to the one directly observed in the host environment. EMEM and DMEM have a standard composition, without expensive and complex components, and are available worldwide at affordable cost. These alternative media can provide researchers with reliable culture conditions for leptospires, facilitating research and its reproducibility, while accelerating discoveries in the field for this neglected disease worldwide.