COS 153-6 - Cloacal Swabs v Feces: Variation and Contamination in microbial samples from Sceloporus lizards

Background/Question/Methods

Microbial communities recovered from feces and cloacal swabs are often used to represent the gut microbiome. These methods are attractive because they are non-destructive, minimally invasive, and allow for repeat sampling of the same individuals and communities over time. However, both methods have their own pitfalls. Fecal communities can be influenced by transient microbes that are not part of the resident microbiome. Different regions of the gut can have unique microbial communities, and the cloaca in particular is under unique selective pressures, as it is the terminus of the gut as well as the site of copulatory intromission and egg laying or birth. We used Illumina high-throughput amplicon sequencing to compare the two sampling methods in Sceloporus virgatus (striped plateau lizard). S. virgatus is an oviparous spiny lizard, with a specialized cloacal microbiome that protects eggs from soil fungus during incubation. We took tissue samples from different regions of the gut and reproductive system, fecal samples, and cloacal swabs (taken both before and after defecation), to determine how well each sampling method represented the gut microbiome and compare the communities recovered from feces and cloacal swabs directly.

Results/Conclusions

In Sceloporus virgatus, we found that cloacal swabs were representative of only certain gut regions, the fecal microbiome did not correspond well to any gut region, and that while the two methods recovered different communities, swabs could be contaminated with fecal microbes. We also found that visual inspection for feces on cloacal swabs was not sufficient to identify contamination. We used these recovered communities to identify high abundance of the Lachnospiraceae family as an indicator of fecal contamination in Sceloporus cloacal swabs. We then developed a method for identification and removal of contaminated samples across three Sceloporus species: S. virgatus, S. occidentalis, and S. jarrovii. These results highlight the importance of method validation. It is imperative to ensure that the sampling method used is representative of the targeted microbial community; in this case, cloacal swabs were representative of the cloacal community, but not other gut regions. Further, important patterns in the data could have been obscured by fecal microbes, which seem to be largely transient and do not establish anywhere along the digestive or reproductive tract. The potential for fecal contamination also offers an explanation for the common finding of extremely variable microbial communities recovered from cloacal swabs.