pLxIS-containing domains are biochemically flexible regulators of interferons and metabolism.

Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.

Undecaprenyl phosphate translocases confer conditional microbial fitness.

The microbial cell wall is essential for maintenance of cell shape and resistance to external stressors1. The primary structural component of the cell wall is peptidoglycan, a glycopolymer with peptide crosslinks located outside of the cell membrane1. Peptidoglycan biosynthesis and structure are responsive to shifting environmental conditions such as pH and salinity2-6, but the mechanisms underlying such adaptations are incompletely understood. Precursors of peptidoglycan and other cell surface glycopolymers are synthesized in the cytoplasm and then delivered across the cell membrane bound to the recyclable lipid carrier undecaprenyl phosphate7 (C55-P, also known as UndP). Here we identify the DUF368-containing and DedA transmembrane protein families as candidate C55-P translocases, filling a critical gap in knowledge of the proteins required for the biogenesis of microbial cell surface polymers. Gram-negative and Gram-positive bacteria lacking their cognate DUF368-containing protein exhibited alkaline-dependent cell wall and viability defects, along with increased cell surface C55-P levels. pH-dependent synthetic genetic interactions between DUF368-containing proteins and DedA family members suggest that C55-P transporter usage is dynamic and modulated by environmental inputs. C55-P transporter activity was required by the cholera pathogen for growth and cell shape maintenance in the intestine. We propose that conditional transporter reliance provides resilience in lipid carrier recycling, bolstering microbial fitness both inside and outside the host.

Reverse transcriptase inhibitors prevent liver abscess formation during Escherichia coli bloodstream infection.

The presence of bacteria in the bloodstream is associated with severe clinical outcomes. In mice, intravenous inoculation of Escherichia coli can lead to the formation of macroscopic abscesses in the liver. Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Liver abscess susceptibility varies widely across strains of mice, but the host factors governing this variation are unknown. Here, we profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. Hypothesizing that ERV-encoded reverse transcriptase may generate cytoplasmic DNA and heighten inflammatory responses, we tested whether nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, leading to a concomitant 100,000-fold reduction in bacterial burden. We provide evidence that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings suggest that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to drive abscess formation. The high efficacy of NRTIs in preventing abscess formation suggests that the consequences of reverse transcription on inflammation should be further examined, particularly in infectious diseases where inflammation drives negative clinical outcomes, such as sepsis.

Temporal and spatial dynamics of Listeria monocytogenes central nervous system infection in mice.

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.

Innate immune responses yield tissue-specific bottlenecks that scale with pathogen dose.

To cause infection, pathogens must overcome bottlenecks imposed by the host immune system. These bottlenecks restrict the inoculum and largely determine whether pathogen exposure results in disease. Infection bottlenecks therefore quantify the effectiveness of immune barriers. Here, using a model of Escherichia coli systemic infection, we identify bottlenecks that tighten or widen with higher inoculum sizes, revealing that the efficacy of innate immune responses can increase or decrease with pathogen dose. We term this concept "dose scaling". During E. coli systemic infection, dose scaling is tissue specific, dependent on the lipopolysaccharide (LPS) receptor TLR4, and can be recapitulated by mimicking high doses with killed bacteria. Scaling therefore depends on sensing of pathogen molecules rather than interactions between the host and live bacteria. We propose that dose scaling quantitatively links innate immunity with infection bottlenecks and is a valuable framework for understanding how the inoculum size governs the outcome of pathogen exposure.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of Escherichia coli leads to bacterial replication within liver abscesses, while other organs such as the spleen clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Abscess susceptibility is heightened in the C57BL lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as 1 d post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4 (Toll-like receptor 4), are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci.

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health.

Increased Listeria monocytogenes Dissemination and Altered Population Dynamics in Muc2-Deficient Mice.

The mucin Muc2 is a major constituent of the mucus layer that covers the intestinal epithelium and creates a barrier between epithelial cells and luminal commensal or pathogenic microorganisms. The Gram-positive foodborne pathogen Listeria monocytogenes can cause enteritis and also disseminate from the intestine to give rise to systemic disease. L. monocytogenes can bind to intestinal Muc2, but the influence of the Muc2 mucin barrier on L. monocytogenes intestinal colonization and systemic dissemination has not been explored. Here, we used an orogastric L. monocytogenes infection model to investigate the role of Muc2 in host defense against L. monocytogenes Compared to wild-type mice, we found that Muc2-/- mice exhibited heightened susceptibility to orogastric challenge with L. monocytogenes, with higher mortality, elevated colonic pathology, and increased pathogen burdens in both the intestinal tract and distal organs. In contrast, L. monocytogenes burdens were equivalent in wild-type and Muc2-/- animals when the pathogen was administered intraperitoneally, suggesting that systemic immune defects related to Muc2 deficiency do not explain the heightened pathogen dissemination observed in oral infections. Using a barcoded L. monocytogenes library to measure intrahost pathogen population dynamics, we found that Muc2-/- animals had larger pathogen founding population sizes in the intestine and distal sites than observed in wild-type animals. Comparisons of barcode frequencies suggested that the colon becomes the major source for seeding the internal organs in Muc2-/- animals. Together, our findings reveal that Muc2 mucin plays a key role in controlling L. monocytogenes colonization, dissemination, and population dynamics.

Bacteriophage Resistance Alters Antibiotic-Mediated Intestinal Expansion of Enterococci.

Enterococcus faecalis is a human intestinal pathobiont with intrinsic and acquired resistance to many antibiotics, including vancomycin. Nature provides a diverse and virtually untapped repertoire of bacterial viruses, or bacteriophages (phages), that could be harnessed to combat multidrug-resistant enterococcal infections. Bacterial phage resistance represents a potential barrier to the implementation of phage therapy, emphasizing the importance of investigating the molecular mechanisms underlying the emergence of phage resistance. Using a cohort of 19 environmental lytic phages with tropism against E. faecalis, we found that these phages require the enterococcal polysaccharide antigen (Epa) for productive infection. Epa is a surface-exposed heteroglycan synthesized by enzymes encoded by both conserved and strain-specific genes. We discovered that exposure to phage selective pressure favors mutation in nonconserved epa genes both in culture and in a mouse model of intestinal colonization. Despite gaining phage resistance, epa mutant strains exhibited a loss of resistance to cell wall-targeting antibiotics. Finally, we show that an E. faecalisepa mutant strain is deficient in intestinal colonization, cannot expand its population upon antibiotic-driven intestinal dysbiosis, and fails to be efficiently transmitted to juvenile mice following birth. This study demonstrates that phage therapy could be used in combination with antibiotics to target enterococci within a dysbiotic microbiota. Enterococci that evade phage therapy by developing resistance may be less fit at colonizing the intestine and sensitized to vancomycin, preventing their overgrowth during antibiotic treatment.

Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

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

Murine models are often used to study the pathogenicity and dissemination of the enteric pathogen Salmonella enterica serovar Typhimurium. Here, we quantified S. Typhimurium population dynamics in mice using the STAMPR analytic pipeline and a highly diverse S. Typhimurium barcoded library containing ~55,000 unique strains distinguishable by genomic barcodes by enumerating S. Typhimurium founding populations and deciphering routes of spread in mice. We found that a severe bottleneck allowed only one in a million cells from an oral inoculum to establish a niche in the intestine. Furthermore, we observed compartmentalization of pathogen populations throughout the intestine, with few barcodes shared between intestinal segments and feces. This severe bottleneck widened and compartmentalization was reduced after streptomycin treatment, suggesting the microbiota plays a key role in restricting the pathogen's colonization and movement within the intestine. Additionally, there was minimal sharing between the intestine and extraintestinal organ populations, indicating dissemination to extraintestinal sites occurs rapidly, before substantial pathogen expansion in the intestine. Bypassing the intestinal bottleneck by inoculating mice via intravenous or intraperitoneal injection revealed that Salmonella re-enters the intestine after establishing niches in extraintestinal sites by at least two distinct pathways. One pathway results in a diverse intestinal population. The other re-seeding pathway is through the bile, where the pathogen is often clonal, leading to clonal intestinal populations and correlates with gallbladder pathology. Together, these findings deepen our understanding of Salmonella population dynamics.

Inducible transposon mutagenesis for genome-scale forward genetics.

Transposon insertion sequencing (Tn-seq) is a powerful method for genome-scale functional genetics in bacteria. However, its effectiveness is often limited by a lack of mutant diversity, caused by either inefficient transposon delivery or stochastic loss of mutants due to population bottlenecks. Here, we introduce "InducTn-seq", which leverages inducible mutagenesis for temporal control of transposition. InducTn-seq generates millions of transposon mutants from a single colony, enabling the sensitive detection of subtle fitness defects and transforming binary classifications of gene essentiality into a quantitative fitness measurement across both essential and non-essential genes. Using a mouse model of infectious colitis, we show that InducTn-seq bypasses a highly restrictive host bottleneck to generate a diverse transposon mutant population from the few cells that initiate infection, revealing the role of oxygen-related metabolic plasticity in pathogenesis. Overall, InducTn-seq overcomes the limitations of traditional Tn-seq, unlocking new possibilities for genome-scale forward genetic screens in bacteria.

Pseudomonas aeruginosa population dynamics in a vancomycin-induced murine model of gastrointestinal carriage.

Pseudomonas aeruginosa is a common nosocomial pathogen and a major cause of morbidity and mortality in hospitalized patients. Multiple reports highlight that P. aeruginosa gastrointestinal colonization may precede systemic infections by this pathogen. Gaining a deeper insight into the dynamics of P. aeruginosa gastrointestinal carriage is an essential step in managing gastrointestinal colonization and could contribute to preventing bacterial transmission and progression to systemic infection. Here, we present a clinically relevant mouse model relying on parenteral vancomycin pretreatment and a single orogastric gavage of a controlled dose of P. aeruginosa. Robust carriage was observed with multiple clinical isolates, and carriage persisted for up to 60 days. Histological and microbiological examination of mice indicated that this model indeed represented carriage and not infection. We then used a barcoded P. aeruginosa library along with the sequence tag-based analysis of microbial populations (STAMPR) analytic pipeline to quantify bacterial population dynamics and bottlenecks during the establishment of the gastrointestinal carriage. Analysis indicated that most of the P. aeruginosa population was rapidly eliminated in the stomach, but the few bacteria that moved to the small intestine and the caecum expanded significantly. Hence, the stomach constitutes a significant barrier against gastrointestinal carriage of P. aeruginosa, which may have clinical implications for hospitalized patients.

Innate immune responses yield tissue-specific bottlenecks that scale with pathogen dose.

To cause infection, pathogens must overcome bottlenecks imposed by the host immune system. These bottlenecks restrict the inoculum and largely determine whether pathogen exposure results in disease. Infection bottlenecks therefore quantify the effectiveness of immune barriers. Here, using a model of Escherichia coli systemic infection, we identify bottlenecks that tighten or widen with higher inoculum sizes, revealing that the efficacy of innate immune responses can increase or decrease with pathogen dose. We term this concept "dose scaling". During E. coli systemic infection, dose scaling is tissue specific, dependent on the LPS receptor TLR4, and can be recapitulated by mimicking high doses with killed bacteria. Scaling is therefore due to sensing of pathogen molecules rather than interactions between the host and live bacteria. We propose that dose scaling quantitatively links innate immunity with infection bottlenecks and is a valuable framework for understanding how the inoculum size governs the outcome of pathogen exposure.

Quantitative dose-response analysis untangles host bottlenecks to enteric infection.

Host bottlenecks prevent many infections before the onset of disease by eliminating invading pathogens. By monitoring the diversity of a barcoded population of the diarrhea causing bacterium Citrobacter rodentium during colonization of its natural host, mice, we determine the number of cells that found the infection by establishing a replicative niche. In female mice the size of the pathogen's founding population scales with dose and is controlled by a severe yet slow-acting bottleneck. Reducing stomach acid or changing host genotype modestly relaxes the bottleneck without breaking the fractional relationship between dose and founders. In contrast, disrupting the microbiota causes the founding population to no longer scale with the size of the inoculum and allows the pathogen to infect at almost any dose, indicating that the microbiota creates the dominant bottleneck. Further, in the absence of competition with the microbiota, the diversity of the pathogen population slowly contracts as the population is overtaken by bacteria having lost the critical virulence island, the locus of enterocyte effacement (LEE). Collectively, our findings reveal that the mechanisms of protection by colonization bottlenecks are reflected in and can be generally defined by the impact of dose on the pathogen's founding population.

CanB is a metabolic mediator of antibiotic resistance in Neisseria gonorrhoeae.

The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments and antibiotics. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2 dependence is attributable to a single substitution in a ß-carbonic anhydrase, CanB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2 dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has affected the acquisition of fluoroquinolone resistance.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of E. coli leads to bacterial replication within liver abscesses while other organs such as the spleen largely clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Susceptibility to liver abscesses is heightened in the C57BL/6 lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as one day post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4, are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Nontoxigenic Vibrio cholerae Challenge Strains for Evaluating Vaccine Efficacy and Inferring Mechanisms of Protection.

Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba O1 V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. Since the primary mechanism of immune protection against cholera is thought to be antibody responses that limit V. cholerae colonization and not the diarrheagenic actions of cholera toxin, these strains were rendered nontoxigenic. In infant mice, the ZChol strains did not cause diarrhea and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. ZChol strains were also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination and passive immunity in the infant mouse model tightens the infection bottleneck without restricting pathogen expansion during intestinal infection. Collectively, our findings suggest that ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera. IMPORTANCE Human challenge studies are a valuable method for testing the efficacy of cholera vaccines. However, challenge studies cannot be performed in countries of cholera endemicity due to safety concerns; also, contemporary pandemic Vibrio cholerae strains are not used in current challenge studies. To facilitate cholera research, we derived nontoxigenic challenge strains of both V. cholerae serotypes from a 2016 clinical isolate from Zambia and demonstrated how they can be used to gauge cholera immunity accurately and safely. These strains were also genetically barcoded, adding the potential for analyses of V. cholerae population dynamics to challenge studies. Preclinical analyses presented here suggest that these strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

Pathogen clonal expansion underlies multiorgan dissemination and organ-specific outcomes during murine systemic infection.

The dissemination of pathogens through blood and their establishment within organs lead to severe clinical outcomes. However, the within-host dynamics that underlie pathogen spread to and clearance from systemic organs remain largely uncharacterized. In animal models of infection, the observed pathogen population results from the combined contributions of bacterial replication, persistence, death, and dissemination, each of which can vary across organs. Quantifying the contribution of each these processes is required to interpret and understand experimental phenotypes. Here, we leveraged STAMPR, a new barcoding framework, to investigate the population dynamics of extraintestinal pathogenic Escherichia coli, a common cause of bacteremia, during murine systemic infection. We show that while bacteria are largely cleared by most organs, organ-specific clearance failures are pervasive and result from dramatic expansions of clones representing less than 0.0001% of the inoculum. Clonal expansion underlies the variability in bacterial burden between animals, and stochastic dissemination of clones profoundly alters the pathogen population structure within organs. Despite variable pathogen expansion events, host bottlenecks are consistent yet highly sensitive to infection variables, including inoculum size and macrophage depletion. We adapted our barcoding methodology to facilitate multiplexed validation of bacterial fitness determinants identified with transposon mutagenesis and confirmed the importance of bacterial hexose metabolism and cell envelope homeostasis pathways for organ-specific pathogen survival. Collectively, our findings provide a comprehensive map of the population biology that underlies bacterial systemic infection and a framework for barcode-based high-resolution mapping of infection dynamics.

Refined Quantification of Infection Bottlenecks and Pathogen Dissemination with STAMPR.

Pathogen population dynamics during infection are critical determinants of infection susceptibility and define patterns of dissemination. However, deciphering these dynamics, particularly founding population sizes in host organs and patterns of dissemination between organs, is difficult because measuring bacterial burden alone is insufficient to observe these patterns. Introduction of allelic diversity into otherwise identical bacteria using DNA barcodes enables sequencing-based measurements of these parameters, in a method known as STAMP (Sequence Tag-based Analysis of Microbial Populations). However, bacteria often undergo unequal expansion within host organs, resulting in marked differences in the frequencies of barcodes in input and output libraries. Here, we show that these differences confound STAMP-based analyses of founding population sizes and dissemination patterns. We present STAMPR, a successor to STAMP, which accounts for such population expansions. Using data from systemic infection of barcoded extraintestinal pathogenic E. coli, we show that this new framework, along with the metrics it yields, enhances the fidelity of measurements of bottlenecks and dissemination patterns. STAMPR was also validated on an independent barcoded Pseudomonas aeruginosa data set, uncovering new patterns of dissemination within the data. This framework (available at https://github.com/hullahalli/stampr_rtisan), when coupled with barcoded data sets, enables a more complete assessment of within-host bacterial population dynamics. IMPORTANCE Barcoded bacteria are often employed to monitor pathogen population dynamics during infection. The accuracy of these measurements is diminished by unequal bacterial expansion rates. Here, we develop computational tools to circumvent this limitation and establish additional metrics that collectively enhance the fidelity of measuring within-host pathogen founding population sizes and dissemination patterns. These new tools will benefit future studies of the dynamics of pathogens and symbionts within their respective hosts and may have additional barcode-based applications beyond host-microbe interactions.