Kazachstania slooffiae is a dimorphic fungus which colonizes the feces and gastrointestinal tract of postweaning pigs. This fungus persists in the gut environment of piglets into adulthood and is implicated in porcine health through microbe-microbe and microbe-host interactions. Here, we report a draft genome sequence for K. slooffiae ABBL.

Low moisture foods (LMFs) have traditionally been recognized as safe for consumption, as most bacteria require higher water content to grow. However, outbreaks due to LMF foods are increasing, and the microbial pathogen Salmonella enterica is frequently implicated. S. enterica can survive in LMFs for years, but few serovars have been studied, and the mechanisms which underlie this longevity are not well understood. Here, we determine that S. enterica serovars S. Tennessee, S. Anatum, and S. Reading but not S. Oranienburg can survive in the ground black pepper for 6 years. S. Reading was not previously associated with any LMF. Using both Illumina and Pacific Biosciences sequencing technologies, we also document changes in the genomes and methylomes of the surviving serovars over this 6-year period. The three serovars acquired a small number of single nucleotide polymorphisms (SNPs) including seven substitutions (four synonymous, two non-synonymous, and one substitution in a non-coding region), and two insertion-deletions. Nine distinct N6-methyladenine (m6A) methylated motifs across the three serovars were identified including five which were previously known, Gm6ATC, CAGm6AG, BATGCm6AT, CRTm6AYN6CTC, and CCm6AN7TGAG, and four novel serovar-specific motifs, GRTm6AN8TTYG, GAm6ACN7GTA, GAA m6ACY, and CAAm6ANCC. Interestingly, the BATGCAT motif was incompletely methylated (35–64% sites across the genome methylated), suggesting a possible role in gene regulation. Furthermore, the number of methylated BATGCm6AT motifs increased after storage in ground black pepper for 6 years from 475 to 657 (S. Tennessee), 366 to 608 (S. Anatum), and 525 to 570 (S. Reading), thus warranting further study as an adaptive mechanism. This is the first long-term assessment of genomic changes in S. enterica in a low moisture environment, and the first study to examine the methylome of any bacteria over a period of years, to our knowledge. These data contribute to our understanding of S. enterica survival in LMFs, and coupled with further studies, will provide the information necessary to design effective interventions which reduce S. enterica in LMFs and maintain a healthy, safe food supply.

The microbiome is an integral part of chicken health and can affect immunity, nutrient utilization, and performance. The role of bacterial microbiota members in host health is relatively well established, but less attention has been paid to fungal members of the gastrointestinal tract (GIT) community. However, human studies indicate that fungi play a critical role in health. Here, we described fungal communities, or mycobiomes, in both the lumen and mucosa of the chicken ileum and cecum from hatch through 14 days of age. We also assessed the effects of delayed access to feed immediately post-hatch (PH) on mycobiome composition, as PH feed delay is commonly associated with poor health performance. Chicken mycobiomes in each of the populations were distinct and changed over time. All mycobiomes were dominated by Gibberella, but Aspergillus, Cladosporium, Sarocladium, Meyerozyma, and Penicillium were also abundant. Relative abundances of some taxa differed significantly over time. In the cecal and ileal lumens, Penicillium was present in extremely low quantities or absent during days one and two and then increased over time. Meyerozyma and Wickerhamomyces also increased over time in luminal sites. In contrast, several highly abundant unclassified fungi decreased after days one and two, highlighting the need for improved understanding of fungal gut biology. Mycobiomes from chicks fed during the first 2 days PH versus those not fed during the first 2 days did not significantly differ, except during days one and two. Similarities observed among mycobiomes of fed and unfed chicks at later timepoints suggest that delays in PH feeding do not have long lasting effects on mycobiome composition. Together, these results provide a foundation for future mycobiome studies, and suggest that negative health and production impacts of delayed feeding are not likely related to the development of fungal populations in the GIT.

IntroductionThe gut microbiome is an integral partner in host health and plays a role in immune development, altered nutrition, and pathogen prevention. The mycobiome (fungal microbiome) is considered part of the rare biosphere but is still a critical component in health. Next generation sequencing has improved our understanding of fungi in the gut, but methodological challenges remain. Biases are introduced during DNA isolation, primer design and choice, polymerase selection, sequencing platform selection, and data analyses, as fungal reference databases are often incomplete or contain erroneous sequences.MethodsHere, we compared the accuracy of taxonomic identifications and abundances from mycobiome analyses which vary among three commonly selected target gene regions (18S, ITS1, or ITS2) and the reference database (UNITE - ITS1, ITS2 and SILVA - 18S). We analyze multiple communities including individual fungal isolates, a mixed mock community created from five common fungal isolates found in weanling piglet feces, a purchased commercial fungal mock community, and piglet fecal samples. In addition, we calculated gene copy numbers for the 18S, ITS1, and ITS2 regions of each of the five isolates from the piglet fecal mock community to determine whether copy number affects abundance estimates. Finally, we determined the abundance of taxa from several iterations of our in-house fecal community to assess the effects of community composition on taxon abundance.ResultsOverall, no marker-database combination consistently outperformed the others. Internal transcribed space markers were slightly superior to 18S in the identification of species in tested communities, but Lichtheimia corymbifera, a common member of piglet gut communities, was not amplified by ITS1 and ITS2 primers. Thus, ITS based abundance estimates of taxa in piglet mock communities were skewed while 18S marker profiles were more accurate. Kazachstania slooffiae displayed the most stable copy numbers (83-85) while L. corymbifera displayed significant variability (90-144) across gene regions.DiscussionThis study underscores the importance of preliminary studies to assess primer combinations and database choice for the mycobiome sample of interest and raises questions regarding the validity of fungal abundance estimates.