Quantification of Salmonella enterica serovar Typhimurium Population Dynamics in Murine Infection Using a Highly Diverse Barcoded Library

Reviewer #1 (Public review):

Hotinger et al. explore the population dynamics of Salmonella enterica serovar Typhimurium in mice using genetically tagged bacteria. In addition to physiological observations, pathology assessments, and CFU measurements, the study emphasizes quantifying host bottleneck sizes that limit Salmonella colonization and dissemination. The authors also investigate the genetic distances between bacterial populations at various infection sites within the host.

Initially, the study confirms that pretreatment with the antibiotic streptomycin before inoculation via orogastric gavage increases the bacterial burden in the gastrointestinal (GI) tract, leading to more severe symptoms and heightened fecal shedding of bacteria. This pretreatment also significantly reduces between-animal variation in bacterial burden and fecal shedding. The authors then calculate founding population sizes across different organs, discovering a severe bottleneck in the intestine, with founding populations reduced by approximately 10^6-fold compared to the inoculum size. Streptomycin pretreatment increases the founding population size and bacterial replication in the GI tract. Moreover, by calculating genetic distances between populations, the authors demonstrate that, in untreated mice, Salmonella populations within the GI tract are genetically dissimilar, suggesting limited exchange between colonization sites. In contrast, streptomycin pretreatment reduces genetic distances, indicating increased exchange.

In extraintestinal organs, the bacterial burden is generally not substantially increased by streptomycin pretreatment, with significant differences observed only in the mesenteric lymph nodes and bile. However, the founding population sizes in these organs are increased. By comparing genetic distances between organs, the authors provide evidence that subpopulations colonizing extraintestinal organs diverge early after infection from those in the GI tract. This hypothesis is further tested by measuring bacterial burden and founding population sizes in the liver and GI tract at 5 and 120 hours post-infection. Additionally, they compare orogastric gavage infection with the less injurious method of infection via drinking, finding similar results for CFUs, founding populations, and genetic distances. These results argue against injuries during gavage as a route of direct infection.

To bypass bottlenecks associated with the GI tract, the authors compare intravenous (IV) and intraperitoneal (IP) routes of infection. They find approximately a 10-fold increase in bacterial burden and founding population size in immune-rich organs with IV/IP routes compared to orogastric gavage in streptomycin-pretreated animals. This difference is interpreted as a result of "extra steps required to reach systemic organs."

While IP and IV routes yield similar results in immune-rich organs, IP infections lead to higher bacterial burdens in nearby sites, such as the pancreas, adipose tissue, and intraperitoneal wash, as well as somewhat increased founding population sizes. The authors correlate these findings with the presence of white lesions in adipose tissue. Genetic distance comparisons reveal that, apart from the spleen and liver, IP infections lead to genetically distinct populations in infected organs, whereas IV infections generally result in higher genetic similarity.

Finally, the authors investigate GI tract reseeding, identifying two distinct routes. They observe that the GI tracts of IP/IV-infected mice are colonized either by a clonal or a diversely tagged bacterial population. In clonally reseeded animals, the genetic distance within the GI tract is very low (often zero) compared to the bile population, which is predominantly clonal or pauciclonal. These animals also display pathological signs, such as cloudy/hardened bile and increased bacterial burden, leading the authors to conclude that the GI tract was reseeded by bacteria from the gallbladder bile. In contrast, animals reseeded by more complex bacterial populations show that bile contributes only a minor fraction of the tags. Given the large founding population size in these animals' GI tracts, which is larger than in orogastrically infected animals, the authors suggest a highly permissive second reseeding route, largely independent of bile. They speculate that this route may involve a reversal of known mechanisms that the pathogen uses to escape from the intestine.

The manuscript presents a substantial body of work that offers a meticulously detailed understanding of the population dynamics of S. Typhimurium in mice. It quantifies the processes shaping the within-host dynamics of this pathogen and provides new insights into its spread, including previously unrecognized dissemination routes. The methodology is appropriate and carefully executed, and the manuscript is well-written, clearly presented, and concise. The authors' conclusions are well-supported by experimental results and thoroughly discussed. This work underscores the power of using highly diverse barcoded pathogens to uncover the within-host population dynamics of infections and will likely inspire further investigations into the molecular mechanisms underlying the bottlenecks and dissemination routes described here.

Major point:

Substantial conclusions in the manuscript rely on genetic distance measurements using the Cavalli-Sforza chord distance. However, it is unclear whether these genetic distance measurements are independent of the founding population size. I would anticipate that in populations with larger founding population sizes, where the relative tag frequencies are closer to those in the inoculum, the genetic distances would appear smaller compared to populations with smaller founding sizes independent of their actual relatedness. This potential dependency could have implications for the interpretation of findings, such as those in Figures 2B and 2D, where antibiotic-pretreated animals consistently exhibit higher founding population sizes and smaller genetic distances compared to untreated animals.

Reviewer #2 (Public review):

In this paper, Hotinger et. al. propose an improved barcoded library system, called STAMPR, to study Salmonella population dynamics during infection. Using this system, the authors demonstrate significant diversity in the colonization of different Salmonella clones (defined by the presence of different barcodes) not only across different organs (liver, spleen, adipose tissues, pancreas, and gall bladder) but also within different compartments of the same gastrointestinal tissue. Additionally, this system revealed that microbiota competition is the major bottleneck in Salmonella intestinal colonization, which can be mitigated by streptomycin treatment. However, this has been demonstrated previously in numerous publications. They also show that there was minimal sharing between populations found in the intestine and those in the other organs. Upon IV and IP infection to bypass the intestinal bottleneck, they were able to demonstrate, using this library, that Salmonella can renter the intestine through two possible routes. One route is essentially the reverse path used to escape the gut, leading to a diverse intestinal population; while the other, through the bile, typically results in a clonal population. Although the authors showed that the STAMPR pipeline improved the ability to identify founder populations and their diversity within the same animal during infections, some of the conclusions appear speculative and not fully supported.

(1) It's particularly interesting how the authors, using this system, demonstrate the dominant role of the microbiota bottleneck in Salmonella colonization and how it is widened by antibiotic treatment (Figure 1). Additionally, the ability to track Salmonella reseeding of the gut from other organs starting with IV and IP injections of the pathogen provides a new tool to study population dynamics (Figure 5). However, I don't think it is possible to argue that the proximal and distal small intestine, Peyer's patches (PPs), cecum, colon, and feces have different founder populations for reasons other than stochastic variations. All the barcoded Salmonella clones have the same fitness and the fact that some are found or expanded in one region of the gastrointestinal tract rather than another likely results from random chance - such as being forced in a specific region of the gut for physical or spatial reasons-and subsequent expansion, rather than any inherent biological cause. For example, some bacteria may randomly adhere to the mucus, some may swim toward the epithelial layer, while others remain in the lumen; all will proliferate in those respective sites. In this way, different founder populations arise based on random localization during movement through the gastrointestinal tract, which is an observation, but it doesn't significantly contribute to understanding pathogen colonization dynamics or pathogenesis. Therefore, I would suggest placing less emphasis on describing these differences or better discussing this aspect, especially in the context of the gastrointestinal tract.

(2) I do think that STAMPR is useful for studying the dynamics of pathogen spread to organs where Salmonella likely resides intracellularly (Figure 3). The observation that the liver is colonized by an early intestinal population, which continues to proliferate at a steady rate throughout the infection, is very interesting and may be due to the unique nature of the organ compared to the mucosal environment. What is the biological relevance during infection? Do the authors observe the same pattern (Figures 3C and G) when normalizing the population data for the spleen and mesenteric lymph nodes (mLN)? If not, what do the authors think is driving this different distribution?

(3) Figure 6: Could the bile pathology be due to increased general bacterial translocation rather than Salmonella colonization specifically? Did the authors check for the presence of other bacteria (potentially also proliferating) in the bile? Do the authors know whether Salmonella's metabolic activity in the bile could be responsible for gallbladder pathology?