Longitudinal human gut microbiome datasets generated using community-level, sequence-based approaches often report a sub-set of long-lived “resident” taxa that rarely, if ever, are lost. This result contrasts with population-level turnover of resident clones on the order of months to years. We hypothesized that the disconnect between these results is due to a relative lack of simultaneous discrimination of the human gut microbiome at both the community and population-levels. Here, we present results of a small, longitudinal cohort study (n = 8 participants) of healthy human adults that identifies static and dynamic members of the gut microbiome at the clone level based on cultivation/genetic discrimination and at the operational taxonomic unit/amplified sequence variant levels based on 16S rRNA sequencing. We provide evidence that there is little “stability” within resident clonal populations of the common gut microbiome bacterial family, Enterobacteriaceae. Given that clones can vary substantially in genome content and that evolutionary processes operate on the population level, these results question the biological relevance of apparent stability at higher taxonomic levels.

Escherichia coli is one of the most well-studied bacterial species, but several significant knowledge gaps remain regarding its ecology and natural history. Specifically, the most important factors influencing its life as a member of the healthy human gut microbiome are either underevaluated or currently unknown. Distinct E. coli population dynamics have been observed over the past century from a handful of temporal studies conducted in healthy human adults. Early studies using serology up to the most recent studies using genotyping and DNA sequencing approaches have all identified long-lived E. coli residents and short-lived transients. This review summarizes these discoveries and other studies that focused on the underlying mechanisms that lead to establishment and maintenance of E. coli residency in healthy human adults. Many fundamental knowledge gaps remain and are highlighted with the hope of facilitating future studies in this exciting research area.

g Clostridium difficile is the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations of C. difficile outbreaks and sporadic cases of disease. The most popular C. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands of C. difficile isolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing of C. difficile pathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified. C lostridium difficile infection (CDI) has emerged as the most frequently encountered nosocomial infection in the UnitedStates (1). The clinical manifestations of CDI range from acute, self-limiting diarrhea to fulminant and sometimes fatal colitis (2). Over the past decade, there has been a doubling of CDI-related discharge diagnoses and a 10-fold increase in CDI-attributable mortality in the United States (3). Because of its clinical importance, a number of C. difficile-typing techniques, including pulsed-field gel electrophoresis (PFGE), restriction endonuclease analysis (REA) typing, and PCR ribotyping, have been implemented to differentiate between C. difficile strains and address important epidemiologic questions.PCR ribotyping is the most commonly cited method for typing C. difficile isolates. This technique quantifies the differences in length between 16S rRNA and 23S rRNA encoding genes at approximately 11 rRNA-encoding operons around the C. difficile genome (4). Like other popular typing techniques (REA and PFGE), PCR ribotyping is gel based, and the data are not easily portable between laboratories. Sequence-based methods, like multilocus sequence type (MLST) and genome sequencing, provide highly portable data but are expensive in comparison to gelbased methods. A significant improvement on traditional PCR ribotyping came with the use of a fluorescently labeled PCR primer and sizing of the resulting amplicons using a Sanger-style sequencer (i.e., fluorescent PCR ribotyping) (5). We developed a similar approach and have applied the method to C. difficile isolates from cases of disease and those that circulate in the community (6-11).Fluorescent PC...

Mutually beneficial interspecific interactions are abundant throughout the natural world, including between microbes. Mutualisms between microbes are critical for everything from human health to global nutrient cycling. Studying model microbial mutualisms in the laboratory enables highly controlled experiments for developing and testing evolutionary and ecological hypotheses. In this review, we begin by describing the tools available for studying model microbial mutualisms. We then outline recent insights that laboratory systems have shed on the evolutionary origins, evolutionary dynamics, and ecological features of microbial mutualism. We touch on gaps in our current understanding of microbial mutualisms, note connections to mutualism in nonmicrobial systems, and call attention to open questions ripe for future study. Expected final online publication date for the Annual Review of Ecology, Evolution, and Systematics, Volume 52 is November 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.