The human gut, teeming with microbes, facilitates the transfer of mobile genetic elements, including plasmids. Plasmids, typically extrachromosomal elements, often enhance host fitness through traits like antibiotic resistance or metabolic functions. However, 'cryptic plasmids' lack clear beneficial functions, are often small and multi-copy, and are challenging to study due to the absence of measurable phenotypes. These plasmids, despite their prevalence, are frequently described as selfish elements or genetic parasites. Cryptic plasmid analyses often focus on monocultures, limiting ecological insights. Recent advancements in metagenomics and plasmid prediction algorithms have provided new opportunities to investigate cryptic plasmids within their natural environment. Previous research characterized over 68,000 plasmids from the human gut, identifying pBI143, a cryptic plasmid prevalent across human populations. This study aims to comprehensively characterize pBI143 using 'omics and experimental approaches, investigating its genetic diversity, host range, transmission, impact on bacterial hosts, and associations with health and disease.

The introduction cites numerous studies on plasmids, focusing on their role in microbial evolution, host fitness, and antibiotic resistance. It highlights the challenge of studying cryptic plasmids due to their lack of obvious beneficial functions and the limitations of traditional monoculture-based studies. The introduction also references the authors' previous work characterizing plasmids from the human gut and sets the stage for a deeper investigation into pBI143.

The methodology section is extensive, detailing various techniques employed. It includes metagenomic assembly and read recruitment using the anvi'o software ecosystem, criteria for pBI143 detection, construction of a plasmid vector with tetQ for transfer assays, qPCR analysis for determining copy numbers, methods for calculating purifying selection and characterizing single nucleotide variants (SNVs), and AlphaFold 2 for structural predictions of MobA. Phylogenetic tree construction was based on ribosomal proteins. Analysis of vertical transmission was performed using metagenomic data from mother-infant pairs. In vitro competition experiments and mouse colonization assays were used to assess the fitness impact of pBI143. The methods for calculating the approximate copy number ratio (ACNR) and details on oxidative stress experiments are also comprehensively described.

pBI143, a cryptic plasmid, is incredibly prevalent (up to 92% of individuals in industrialized countries) and abundant in human guts, surpassing crAssphage in copy number. Three distinct pBI143 versions exist, differing primarily in the repA gene. Strong purifying selection acts on pBI143 genes (low dN/dS values). pBI143 exhibits monoclonality within individuals, likely due to priority effects and often vertical transmission from mother to infant. pBI143 is hosted by a wide range of Bacteroidales species, and it can transfer between them. In vivo studies show that pBI143 doesn't significantly affect bacterial host fitness, although it can transiently acquire additional genes. pBI143 serves as a sensitive biomarker for human fecal contamination and shows increased copy number under oxidative stress conditions, such as in inflammatory bowel disease (IBD), as shown by a 3.72 times larger geometric mean of the ACNR in IBD compared to healthy individuals.

The study's findings challenge the traditional understanding of cryptic plasmids as mere parasites. The high prevalence and conservation of pBI143, coupled with its ability to acquire additional genes and respond to stress, suggest a more complex role than previously thought. The strong purifying selection suggests pBI143 provides some yet-unknown benefit to its host, or its highly efficient transmission counteracts the cost of its maintenance. The significant positive correlation between pBI143 and its hosts does not allow to distinguish whether this is a benefit to the host or the cryptic plasmid acts as a genetic hitchhiker. The practical applications, such as detecting fecal contamination and as a potential biomarker for IBD, emphasize the significance of this study.

This research revealed the remarkable prevalence and ecological success of pBI143, a cryptic plasmid that was previously overlooked. Its abundance and conservation, alongside its potential as a biomarker for IBD and for identifying fecal contamination, highlight the importance of studying seemingly inconsequential elements of the microbiome. Future research could focus on the unknown functions of pBI143, its impact on host health beyond IBD, and the mechanisms driving its successful adaptation across diverse environments.

The study relies on publicly available metagenomic data, which may introduce biases related to sample collection, sequencing, and bioinformatics processing. The approximate copy number ratio (ACNR) calculation relies on assumptions that may not perfectly reflect the true copy number in vivo. The in vivo experiments were limited in scope. Further research with larger datasets and more targeted experiments are required to corroborate the findings and fully elucidate the role of pBI143.