New perspectives on viable microbial communities in low-biomass cleanroom environments

Abstract

The advent of phylogenetic DNA microarrays and high-throughput pyrosequencing technologies has dramatically increased the resolution and accuracy of detection of distinct microbial lineages in mixed microbial assemblages. Despite an expanding array of approaches for detecting microbes in a given sample, rapid and robust means of assessing the differential viability of these cells, as a function of phylogenetic lineage, remain elusive. In this study, pre-PCR propidium monoazide (PMA) treatment was coupled with downstream pyrosequencing and PhyloChip DNA microarray analyses to better understand the frequency, diversity and distribution of viable bacteria in spacecraft assembly cleanrooms. Sample fractions not treated with PMA, which were indicative of the presence of both live and dead cells, yielded a great abundance of highly diverse bacterial pyrosequences. In contrast, only 1% to 10% of all of the pyrosequencing reads, arising from a few robust bacterial lineages, originated from sample fractions that had been pre-treated with PMA. The results of PhyloChip analyses of PMA-treated and -untreated sample fractions were in agreement with those of pyrosequencing. The viable bacterial population detected in cleanrooms devoid of spacecraft hardware was far more diverse than that observed in cleanrooms that housed mission-critical spacecraft hardware. The latter was dominated by hardy, robust organisms previously reported to survive in oligotrophic cleanroom environments. Presented here are the findings of the first ever comprehensive effort to assess the viability of cells in low-biomass environmental samples, and correlate differential viability with phylogenetic affiliation.

Resilient microorganisms in dust samples of the International Space Station—survival of the adaptation specialists

“…Notably, Bradyrhizobium sp. (Accession number AY599676), now classified as B. erythrophlei , was also recently observed in propidium monoazide treated samples of spacecraft assembly facilities [20]. Bradyrhizobium signatures were reported in high abundance even in intensive care units and hospital biofilms [46, 47].…”

“…One aliquot was directly frozen at −80 °C, and one was treated with an end concentration of 50 μM propidium monoazide (PMA), to block free DNA of dead cells from downstream applications [20] before freezing.…”

Effects of Propidium Monoazide (PMA) Treatment on Mycobiome and Bacteriome Analysis of Cystic Fibrosis Airways during Exacerbation

“…Several other studies using HTS compared the bacterial profiles of PMA-treated and untreated environmental samples and showed significant changes in the bacteriome structure [11–14]. Whereas Vaishampayan and colleagues [12] demonstrated a difference in the bacteriome structure of environmental samples with and without PMA-pretreatment using PCA, we found both PMA-treated and untreated samples situated virtually next to each other (Fig 4B), in agreement with Exterkate and colleagues’ results, studying oral cavity samples [14]. …”

Reagent contamination can critically impact sequence-based microbiome analyses

“…A number of methods have been devised to treat reagents in order to reduce potential contamination, including: gamma 45 or UV radiation 13,46–48 ; DNase treatment 10,13,47,49–51 restriction digests 10,13,47,52,53 caesium chloride density gradient centrifugation 10 , and DNA intercalation and crosslinking with 8-methoxypsoralen 47,54 propidium monoazide 55 or ethidium monoazide 56,57 . However tests of these methods show varying levels of success.…”