Desiccation caused DNA damage in Mycobacterium tuberculosis and increased the frequency of rifampin-resistant bacteria recovered in laboratory testing, according to a study published in Nature Microbiology.
The findings suggest that drying during aerosol formation may influence bacterial genetic diversity and the types of resistance mutations that persist during transmission. However, the study did not demonstrate that desiccation produces rifampin resistance in patients or directly increases the transmission of resistant tuberculosis.
Researchers developed a filter-based laboratory system that exposed M tuberculosis to controlled humidity. The model was intended to reproduce the loss of water that occurs when respiratory droplets become airborne.
Drying reduced the number of bacteria that could immediately grow on culture media. Some apparently nonculturable bacteria remained viable, however, and growth could largely be restored by placing them in nutrient-rich liquid media before plating.
Transcriptomic and metabolomic analyses showed that desiccation produced a distinct bacterial stress response. Reactive oxygen species increased over time, accompanied by oxidative damage, lipid oxidation, and double-stranded DNA breaks. Genes involved in oxidative stress and DNA repair were also activated.
When rifampin resistance was used as a marker of genetic mutation, desiccated bacteria produced rifampin-resistant colonies at a frequency 50 to 100 times higher than bacteria maintained in saline. This increase disappeared when the dried bacteria were allowed to recover in nutrient-rich media before susceptibility testing, indicating that recovery conditions affected the observed resistance frequency.
The researchers also examined mfd, a gene involved in transcription-coupled DNA repair. Silencing mfd reduced the frequency of rifampin resistance following desiccation and changed the mutations found in rpoB, which encodes the bacterial RNA polymerase targeted by rifampin.
With normal mfd activity, the clinically common rpoB S450L mutation predominated. When mfd was silenced, S450L was largely replaced by H445Y, another established resistance-associated mutation. Silencing mfd also reduced the survival of bacteria carrying S450L during drying and rehydration.
The team compared these findings with 51,229 clinical M tuberculosis whole-genome sequences. Among isolates with nonsynonymous mfd mutations, H445Y was more common relative to S450L than it was across the complete rifampin-resistant data set. A similar pattern appeared in lineage 1 strains carrying an ancestral mfd variant.
The study highlights the relationship between bacterial physiology, culturability, and resistance-associated genotypes. Molecular assays commonly identify rifampin resistance by detecting mutations within the rifampin resistance-determining region of rpoB. The findings reinforce the importance of recognizing more than one resistance-associated allele and of investigating genotype-phenotype discordance with culture-based susceptibility testing or broader sequencing when appropriate.
The work used laboratory strains and retrospective genomic data. It did not directly observe the development of resistance during aerosol transmission, and the degree of desiccation experienced by bacteria during human transmission remains unknown. The effects of clinical specimen drying or rehydration on routine diagnostic recovery were not tested.
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