A strong priority effect in the assembly of a specialized insect-microbe symbiosis.

Specialized host-microbe symbioses are ecological communities, whose composition is shaped by various processes. Microbial community assembly in these symbioses is determined in part by interactions between taxa that colonize ecological niches available within habitat patches. The outcomes of these interactions, and by extension the trajectory of community assembly, can display priority effects-dependency on the order in which taxa first occupy these niches. The underlying mechanisms of these phenomena vary from system to system and are often not well resolved. Here, we characterize priority effects in colonization of the squash bug (Anasa tristis) by bacterial symbionts from the genus Caballeronia, using pairs of strains that are known to strongly compete during host colonization, as well as strains that are isogenic and thus functionally identical. By introducing symbiont strains into individual bugs in a sequential manner, we show that within-host populations established by the first colonist are extremely resistant to invasion, regardless of strain identity and competitive interactions. By knocking down the population of an initial colonist with antibiotics, we further show that colonization success by the second symbiont is still diminished even when space in the symbiotic organ is available and ostensibly accessible for colonization. We speculate that resident symbionts exclude subsequent infections by manipulating the host environment, partially but not exclusively by eliciting tissue remodeling of the symbiont organ. IMPORTANCE: Host-associated microbial communities underpin critical ecosystem processes and human health, and their ability to do so is determined in turn by the various processes that shape their composition. While selection deterministically acts on competing genotypes and species during community assembly, the manner by which selection determines the trajectory of community assembly can differ depending on the sequence by which taxa are established within that community. We document this phenomenon, known as a priority effect, during experimental colonization of a North American insect pest, the squash bug Anasa tristis, by its betaproteobacterial symbionts in the genus Caballeronia. Our study demonstrates how stark, strain-level variation can emerge in specialized host-microbe symbioses simply through differences in the order by which strains colonize the host. Understanding the mechanistic drivers of community structure in host-associated microbiomes can highlight both pitfalls and opportunities for the engineering of these communities and their constituent taxa for societal benefit.

The dynamics of Staphylococcal infection and their treatment with antibiotics and bacteriophage in the Galleria mellonella model system.

Critical to our understanding of infections and their treatment is the role the innate immune system plays in controlling bacterial pathogens. Nevertheless, many in vivo systems are made or modified such that they do not have an innate immune response. Use of these systems denies the opportunity to examine the synergy between the immune system and antimicrobial agents. In this study we demonstrate that the larva of Galleria mellonella is an effective in vivo model for the study of the population and evolutionary biology of bacterial infections and their treatment. To do this we test three hypotheses concerning the role of the innate immune system during infection. We show: i) sufficiently high densities of bacteria are capable of saturating the innate immune system, ii) bacteriostatic drugs and bacteriophages are as effective as bactericidal antibiotics in preventing mortality and controlling bacterial densities, and iii) minority populations of bacteria resistant to a treating antibiotic will not ascend. Using a highly virulent strain of Staphylococcus aureus and a mathematical computer-simulation model, we further explore how the dynamics of the infection within the short term determine the ultimate infection outcome. We find that excess immune activation in response to high densities of bacteria leads to a strong but short-lived immune response which ultimately results in a high degree of mortality. Overall, our findings illustrate the utility of the G. mellonella model system in conjunction with established in vivo models in studying infectious disease progression and treatment.

Maximum likelihood estimators for colony-forming units.

Measuring the abundance of microbes in a sample is a common procedure with a long history, but best practices are not well-conserved across microbiological fields. Serial dilution methods are commonly used to dilute bacterial cultures to produce countable numbers of colonies, and from these counts, to infer bacterial concentrations measured in colony-forming units (CFUs). The most common methods to generate data for CFU point estimates involve plating bacteria on (or in) a solid growth medium and counting their resulting colonies or counting the number of tubes at a given dilution that have growth. Traditionally, these types of data have been analyzed separately using different analytic methods. Here, we build a direct correspondence between these approaches, which allows one to extend the use of the most probable number method from the liquid tubes experiments, for which it was developed, to the growth plates by viewing colony-sized patches of a plate as equivalent to individual tubes. We also discuss how to combine measurements taken at different dilutions, and we review several ways of analyzing colony counts, including the Poisson and truncated Poisson methods. We test all point estimate methods computationally using simulated data. For all methods, we discuss their relevant error bounds, assumptions, strengths, and weaknesses. We provide an online calculator for these estimators.Estimation of the number of microbes in a sample is an important problem with a long history. Yet common practices, such as combining results from different measurements, remain sub-optimal. We provide a comparison of methods for estimating abundance of microbes and detail a mapping between different methods, which allows to extend their range of applicability. This mapping enables higher precision estimates of colony-forming units (CFUs) using the same data already collected for traditional CFU estimation methods. Furthermore, we provide recommendations for how to combine measurements of colony counts taken across dilutions, correcting several misconceptions in the literature.

Ecological drift during colonization drives within-host and between-host heterogeneity in an animal-associated symbiont.

Specialized host-microbe symbioses canonically show greater diversity than expected from simple models, both at the population level and within individual hosts. To understand how this heterogeneity arises, we utilize the squash bug, Anasa tristis, and its bacterial symbionts in the genus Caballeronia. We modulate symbiont bottleneck size and inoculum composition during colonization to demonstrate the significance of ecological drift, the noisy fluctuations in community composition due to demographic stochasticity. Consistent with predictions from the neutral theory of biodiversity, we found that ecological drift alone can account for heterogeneity in symbiont community composition between hosts, even when 2 strains are nearly genetically identical. When acting on competing strains, ecological drift can maintain symbiont genetic diversity among different hosts by stochastically determining the dominant strain within each host. Finally, ecological drift mediates heterogeneity in isogenic symbiont populations even within a single host, along a consistent gradient running the anterior-posterior axis of the symbiotic organ. Our results demonstrate that symbiont population structure across scales does not necessarily require host-mediated selection, as it can emerge as a result of ecological drift acting on both isogenic and unrelated competitors. Our findings illuminate the processes that might affect symbiont transmission, coinfection, and population structure in nature, which can drive the evolution of host-microbe symbioses and microbe-microbe interactions within host-associated microbiomes.

Parallel evolution of alternate morphotypes of Chryseobacterium gleum during experimental evolution with Caenorhabditis elegans.

Microbial evolution within polymicrobial communities is a complex process. Here, we report within-species diversification within multispecies microbial communities during experimental evolution with the nematode Caenorhabditis elegans. We describe morphological diversity in the target species Chryseobacterium gleum, which developed a novel colony morphotype in a small number of replicate communities. Alternate morphotypes coexisted with original morphotypes in communities, as well as in single-species experiments using evolved isolates. We found that the original and alternate morphotypes differed in motility and in spatial expansion in the presence of C. elegans. This study provides insight into the emergence and maintenance of intraspecies diversity in the context of microbial communities.

Enhancing nutritional niche and host defenses by modifying the gut microbiome.

The gut microbiome is essential for processing complex food compounds and synthesizing nutrients that the host cannot digest or produce, respectively. New model systems are needed to study how the metabolic capacity provided by the gut microbiome impacts the nutritional status of the host, and to explore possibilities for altering host metabolic capacity via the microbiome. Here, we colonized the nematode Caenorhabditis elegans gut with cellulolytic bacteria that enabled C. elegans to utilize cellulose, an otherwise indigestible substrate, as a carbon source. Cellulolytic bacteria as a community component in the worm gut can also support additional bacterial species with specialized roles, which we demonstrate by using Lactobacillus plantarum to protect C. elegans against Salmonella enterica infection. This work shows that engineered microbiome communities can be used to endow host organisms with novel functions, such as the ability to utilize alternate nutrient sources or to better fight pathogenic bacteria.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.