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ABSTRACT Gram-negative facultative anaerobes often cause bacteremia, a systemic infection associated with severe clinical outcomes. ArcAB, a two-component regulatory system that represses aerobic respiration, is a key mediator of metabolic adaptation for such bacteria. Using targeted mutational analysis informed by global genetic screens, we identified the arcA gene as promoting fitness of Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens but not Escherichia coli in a murine model of bacteremia. arcA mutants exhibit a dysregulated response to changes in oxygen availability, iron limitation, and membrane perturbations, which bacterial cells experience during infection. The genetic response of the arcA mutants to the cationic antimicrobial peptide polymyxin B supports an expanded role for ArcA as an activator in response to membrane damage. ArcA function is linked to electron transport chain activity based on its response to proton motive force uncoupling by carbonylcyanide-m-chlorophenylhydrazone (CCCP). Differences in lactate, acetate, and lactate dehydrogenase activity between arcA mutant and wild-type cells following CCCP treatment support an ArcA-mediated shift to fermentation independent of oxygen availability. This study highlights the semi-conserved role of ArcA during bacteremia and consolidates infection phenotypes into a comprehensive model based on respiratory activity. IMPORTANCE Infections of the bloodstream are life-threatening and can result in sepsis. Gram-negative bacteria cause a significant portion of bloodstream infections, which is also referred to as bacteremia. The long-term goal of our work is to understand how such bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while countering the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

ABSTRACT Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

ABSTRACT Ordered transposon libraries are a valuable resource for many bacterial species, especially those with difficult methods for generating targeted genetic mutations. Here, we present the construction of an ordered transposon library for the bacterial urinary tract pathogen Proteus mirabilis strain HI4320. This library will facilitate future studies into P. mirabilis biology. For large experimental screens, it may be used to overcome bottleneck constraints and avoid biased outcomes resulting from gene length. For smaller studies, the library allows sidestepping the laborious construction of single targeted mutants. This library, containing 18,432 wells, was condensed into a smaller library containing 1,728 mutants. Each selected mutant had a single transposon insertion in an open reading frame, covering 45% of predicted genes encoded by P. mirabilis HI4320. This coverage was lower than expected and was due both to library wells with no mapped insertions and a surprisingly high proportion of mixed clones and multiple transposon insertion events. We offer recommendations for improving future library construction and suggestions for how to use this P. mirabilis library resource. IMPORTANCE Ordered libraries facilitate large genetic screens by guaranteeing high genomic coverage with a minimal number of mutants, and they can save time and effort by reducing the need to construct targeted mutations. This resource is now available for P. mirabilis, a common and complicating agent of catheter-associated urinary tract infection. We also present obstacles encountered during library construction with the goal to aid others who would like to construct ordered transposon libraries in other species.

ABSTRACT More than half of women will experience a urinary tract infection (UTI), with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if HM7 possesses unidentified iron acquisition systems, we performed RNA sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is enriched in UPEC isolates compared to fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔfecA/ΔentB) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

ABSTRACT CpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.

ABSTRACT Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated

ABSTRACT Uropathogenic Escherichia coli (UPEC) is the primary causative agent of uncomplicated urinary tract infections (UTIs). UPEC fitness and virulence determinants have been evaluated in a variety of laboratory settings, including a well-established mouse model of UTI. However, the extent to which bacterial physiologies differ between experimental models and human infections remains largely understudied. To address this important issue, we compared the transcriptomes of three different UPEC isolates in human infection and under a variety of laboratory conditions, including LB culture, filter-sterilized urine culture, and the UTI mouse model. We observed high correlation in gene expression between the mouse model and human infection in all three strains examined (Pearson correlation coefficients of 0.86 to 0.87). Only 175 of 3,266 (5.4%) genes shared by all three strains had significantly different expression levels, with the majority of them (145 genes) downregulated in patients. Importantly, gene expression levels of both canonical virulence factors and metabolic machinery were highly similar between the mouse model and human infection, while the in vitro conditions displayed more substantial differences. Interestingly, comparison of gene expression between the mouse model and human infection hinted at differences in bladder oxygenation as well as nutrient composition. In summary, our work strongly validates the continued use of this mouse model for the study of the pathogenesis of human UTI. IMPORTANCE Different experimental models have been used to study UPEC pathogenesis, including in vitro cultures in different media, tissue culture, and mouse models of infection. The last is especially important since it allows evaluation of mechanisms of pathogenesis and potential therapeutic strategies against UPEC. Bacterial physiology is greatly shaped by environment, and it is therefore critical to understand how closely bacterial physiology in any experimental model relates to human infection. In this study, we found strong correlation in bacterial gene expression between the mouse model and human UTI using identical strains, suggesting that the mouse model accurately mimics human infection, definitively supporting its continued use in UTI research.

ABSTRACT Providencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infection (CAUTI), and yet literature describing the molecular mechanisms of its pathogenesis is limited. To identify factors important for colonization during single-species infection and during polymicrobial infection with a common cocolonizer, Proteus mirabilis, we created a saturating library of ∼50,000 transposon mutants and conducted transposon insertion site sequencing (Tn-Seq) in a murine model of CAUTI. P. stuartii strain BE2467 carries 4,398 genes, 521 of which were identified as essential for growth in laboratory medium and therefore could not be assessed for contribution to infection. Using an input/output fold change cutoff value of 20 and P values of

ABSTRACT Urinary tract infections (UTI) affect half of all women at least once during their lifetime. The rise in the numbers of extended-spectrum beta-lactamase-producing strains and the potential for carbapenem resistance within uropathogenic Escherichia coli (UPEC), the most common causative agent of UTI, create an urgent need for vaccine development. Intranasal immunization of mice with UPEC outer membrane iron receptors FyuA, Hma, IreA, and IutA, conjugated to cholera toxin, provides protection in the bladder or kidneys under conditions of challenge with UPEC strain CFT073 or strain 536. On the basis of these data, we sought to optimize the vaccination route (intramuscular, intranasal, or subcutaneous) in combination with adjuvants suitable for human use, including aluminum hydroxide gel (alum), monophosphoryl lipid A (MPLA), unmethylated CpG synthetic oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (polyIC), and mutated heat-labile E. coli enterotoxin (dmLT). Mice intranasally vaccinated with dmLT-IutA and dmLT-Hma displayed significant reductions in bladder colonization (86-fold and 32-fold, respectively), with 40% to 42% of mice having no detectable CFU. Intranasal vaccination of mice with CpG-IutA and polyIC-IutA significantly reduced kidney colonization (131-fold) and urine CFU (22-fold), respectively. dmLT generated the most consistently robust antibody response in intranasally immunized mice, while MPLA and alum produced greater concentrations of antigen-specific serum IgG with intramuscular immunization. On the basis of these results, we conclude that intranasal administration of Hma or IutA formulated with dmLT adjuvant provides the greatest protection from UPEC UTI. This report advances our progress toward a vaccine against uncomplicated UTI, which will significantly improve the quality of life for women burdened by recurrent UTI and enable better antibiotic stewardship. IMPORTANCE Urinary tract infections (UTI) are among the most common bacterial infection in humans, affecting half of all women at least once during their lifetimes. The rise in antibiotic resistance and health care costs emphasizes the need to develop a vaccine against the most common UTI pathogen, Escherichia coli. Vaccinating mice intranasally with a detoxified heat-labile enterotoxin and two surface-exposed receptors, Hma or IutA, significantly reduced bacterial burden in the bladder. This work highlights progress in the development of a UTI vaccine formulated with adjuvants suitable for human use and antigens that encode outer membrane iron receptors required for infection in the iron-limited urinary tract.

Abstract Background Tn-Seq is a high throughput technique for analysis of transposon mutant libraries to determine conditional essentiality of a gene under an experimental condition. A special feature of the Tn-seq data is that multiple mutants in a gene provides independent evidence to prioritize that gene as being essential. The existing methods do not account for this feature or rely on a high-density transposon library. Moreover, these methods are unable to accommodate complex designs. Results The method proposed here is specifically designed for the analysis of Tn-Seq data. It utilizes two steps to estimate the conditional essentiality for each gene in the genome. First, it collects evidence of conditional essentiality for each insertion by comparing read counts of that insertion between conditions. Second, it combines insertion-level evidence for the corresponding gene. It deals with data from both low- and high-density transposon libraries and accommodates complex designs. Moreover, it is very fast to implement. The performance of the proposed method was tested on simulated data and experimental Tn-Seq data from Serratia marcescens transposon mutant library used to identify genes that contribute to fitness in a murine model of infection. Conclusion We describe a new, efficient method for identifying conditionally essential genes in Tn-Seq experiments with high detection sensitivity and specificity. It is implemented as TnseqDiff function in R package Tnseq and can be installed from the Comprehensive R Archive Network, CRAN.

ABSTRACT Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens. Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism. IMPORTANCE Serratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens. The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

ABSTRACT Antibiotic-resistant Acinetobacter baumannii is increasingly recognized as a cause of difficult-to-treat nosocomial infections, including pneumonia, wound infections, and bacteremia. Previous studies have demonstrated that the metalloprotease CpaA contributes to virulence and prolongs clotting time when added to human plasma as measured by the activated partial thromboplastin time (aPTT) assay. Here, we show that CpaA interferes with the intrinsic coagulation pathway, also called the contact activation system, in human as well as murine plasma, but has no discernible effect on the extrinsic pathway. By utilizing a modified aPTT assay, we demonstrate that coagulation factor XII (fXII) is a target of CpaA. In addition, we map the cleavage by CpaA to two positions, 279-280 and 308-309, within the highly glycosylated proline-rich region of human fXII, and show that cleavage at the 308-309 site is responsible for inactivation of fXII. At both sites, cleavage occurs between proline and an O-linked glycosylated threonine, and deglycosylation of fXII prevents cleavage by CpaA. Consistent with this, mutant fXII (fXII-Thr309Lys) from patients with hereditary angioedema type III (HAEIII) is protected from CpaA inactivation. This raises the possibility that individuals with HAEIII who harbor this mutation may be partially protected from A. baumannii infection if CpaA contributes to human disease. By inactivating fXII, CpaA may attenuate important antimicrobial defense mechanisms such as intravascular thrombus formation, thus allowing A. baumannii to disseminate. IMPORTANCE Ventilator-associated pneumonia and catheter-related bacteremia are the most common and severe infections caused by Acinetobacter baumannii. Besides the capsule, lipopolysaccharides, and the outer membrane porin OmpA, little is known about the contribution of secreted proteins to A. baumannii survival in vivo. Here we focus on CpaA, a potentially recently acquired virulence factor that inhibits blood coagulation in vitro. We identify coagulation factor XII as a target of CpaA, map the cleavage sites, and show that glycosylation is a prerequisite for CpaA-mediated inactivation of factor XII. We propose adding CpaA to a small, but growing list of bacterial proteases that are specific for highly glycosylated components of the host defense system.

ABSTRACT Uropathogenic Escherichia coli strains utilize a variety of adherence factors that assist in colonization of the host urinary tract. TosA (type one secretion A) is a nonfimbrial adhesin that is predominately expressed during murine urinary tract infection (UTI), binds to kidney epithelial cells, and promotes survival during invasive infections. The tosRCBDAEF operon encodes the secretory machinery necessary for TosA localization to the E. coli cell surface, as well as the transcriptional regulator TosR. TosR binds upstream of the tos operon and in a concentration-dependent manner either induces or represses tosA expression. TosR is a member of the PapB family of fimbrial regulators that can participate in cross talk between fimbrial operons. TosR also binds upstream of the pap operon and suppresses PapA production. However, the scope of TosR-mediated cross talk is understudied and may be underestimated. To quantify the global effects of TosR-mediated regulation on the E. coli CFT073 genome, we induced expression of tosR, collected mRNA, and performed high-throughput RNA sequencing (RNA-Seq). These findings show that production of TosR affected the expression of genes involved with adhesins, including P, F1C, and Auf fimbriae, nitrate-nitrite transport, microcin secretion, and biofilm formation. IMPORTANCE Uropathogenic E. coli strains cause the majority of UTIs, which are the second most common bacterial infection in humans. During a UTI, bacteria adhere to cells within the urinary tract, using a number of different fimbrial and nonfimbrial adhesins. Biofilms can also develop on the surfaces of catheters, resulting in complications such as blockage. In this work, we further characterized the regulator TosR, which links both adhesin production and biofilm formation and likely plays a crucial function during UTI and disseminated infection.

ABSTRACT Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo. Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo, matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization.

Nosocomial pathogens that develop multidrug resistance present an increasing problem for healthcare facilities. Due to its rapid rise in antibiotic resistance, Acinetobacter baumannii is one of the most concerning gram-negative species. A. baumannii typically infects immune compromised individuals resulting in a variety of outcomes, including pneumonia and bacteremia. Using a murine model for bacteremia, we have previously shown that the type II secretion system (T2SS) contributes to in vivo fitness of A. baumannii. Here, we provide support for a role of the T2SS in protecting A. baumannii from human complement as deletion of the T2SS gene gspD resulted in a 100-fold reduction in surviving cells when incubated with human serum. This effect was abrogated in the absence of Factor B, a component of the alternative pathway of complement activation, indicating that the T2SS protects A. baumannii against the alternative complement pathway. Because inactivation of the T2SS results in loss of secretion of multiple enzymes, reduced in vivo fitness, and increased sensitivity to human complement, the T2SS may be a suitable target for therapeutic intervention. Accordingly, we developed and optimized a whole-cell high-throughput screening (HTS) assay based on secreted lipase activity to identify small molecule inhibitors of the T2SS. We tested the reproducibility of our assay using a 6,400-compound library. With small variation within controls and a dynamic range between positive and negative controls, the assay had a z-factor of 0.65, establishing its suitability for HTS. Our screen identified the lipase inhibitors Orlistat and Ebelactone B demonstrating the specificity of the assay. To eliminate inhibitors of lipase activity and lipase expression, two counter assays were developed and optimized. By implementing these assays, all seven tricyclic antidepressants present in the library were found to be inhibitors of the lipase, highlighting the potential of identifying alternative targets for approved pharmaceuticals. Although no T2SS inhibitor was identified among the compounds that reduced lipase activity by ≥30%, our small proof-of-concept pilot study indicates that the HTS regimen is simple, reproducible, and specific and that it can be used to screen larger libraries for the identification of T2SS inhibitors that may be developed into novel A. baumannii therapeutics.

ABSTRACT Serratia marcescens is an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of the Enterobacteriaceae family, the genetic factors that facilitate Serratia proliferation within the mammalian host are less well defined. An in vivo screen of transposon insertion mutants identified 212 S. marcescens fitness genes that contribute to bacterial survival in a murine model of bloodstream infection. Among those identified, 11 genes were located within an 18-gene cluster encoding predicted extracellular polysaccharide biosynthesis proteins. A mutation in the wzx gene contained within this locus conferred a loss of fitness in competition infections with the wild-type strain and a reduction in extracellular uronic acids correlating with capsule loss. A second gene, pgm, encoding a phosphoglucomutase exhibited similar capsule-deficient phenotypes, linking central glucose metabolism with capsule production and fitness of Serratia during mammalian infection. Further evidence of the importance of central metabolism was obtained with a pfkA glycolytic mutant that demonstrated reduced replication in human serum and during murine infection. An MgtB magnesium transporter homolog was also among the fitness factors identified, and an S. marcescens mgtB mutant exhibited decreased growth in defined medium containing low concentrations of magnesium and was outcompeted ~10-fold by wild-type bacteria in mice. Together, these newly identified genes provide a more complete understanding of the specific requirements for S. marcescens survival in the mammalian host and provide a framework for further investigation of the means by which S. marcescens causes opportunistic infections. IMPORTANCE Serratia marcescens is a remarkably prolific organism that replicates in diverse environments, including as an opportunistic pathogen in human bacteremia. The genetic requirements for S. marcescens survival in the mammalian bloodstream were defined in this work by transposon insertion sequencing. In total, 212 genes that contribute to bacterial fitness were identified. When sorted via biological function, two of the major fitness categories identified herein were genes encoding capsule polysaccharide biogenesis functions and genes involved in glucose utilization. Further investigation determined that certain glucose metabolism fitness genes are also important for the generation of extracellular polysaccharides. Together, these results identify critical biological processes that allow S. marcescens to colonize the mammalian bloodstream.

Urinary tract infection (UTI) is an extremely common infectious disease. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Asymptomatic bacteriuric E. coli (ABEC) strains successfully colonize the urinary tract resulting in asymptomatic bacteriuria (ABU) and do not induce symptoms associated with UTI. Oxylipids are key signaling molecules involved in inflammation. Based on the distinct clinical outcomes of E. coli colonization, we hypothesized that UPEC triggers the production of predominantly proinflammatory oxylipids and ABEC leads to production of primarily anti-inflammatory or proresolving oxylipids in the urinary tract. We performed quantitative detection of 39 oxylipid mediators with proinflammatory, anti-inflammatory, and proresolving properties, during UTI and ABU caused by genetically distinct E. coli strains in the murine urinary bladder. Our results reveal that infection with UPEC causes an increased accumulation of proinflammatory oxylipids as early as 6 h postinoculation, compared to controls. To the contrary, ABEC colonization leads to decreased accumulation of proinflammatory oxylipids at the early time point compared to UPEC infection but does not affect the level of proresolving oxylipids. This report represents the first comprehensive investigation on the oxylipidome during benign ABEC colonization observed in ABU and acute inflammation triggered by UPEC leading to UTI.

ABSTRACT Acinetobacter baumannii is emerging as a leading global multiple-antibiotic-resistant nosocomial pathogen. The identity of genes essential for pathogenesis in a mammalian host remains largely unknown. Using transposon-directed insertion-site sequencing (TraDIS), we identified A. baumannii genes involved in bacterial survival in a leukopenic mouse model of bloodstream infection. Mice were inoculated with a pooled transposon mutant library derived from 109,000 mutants, and TraDIS was used to map transposon insertion sites in the genomes of bacteria in the inoculum and of bacteria recovered from mouse spleens. Unique transposon insertion sites were mapped and used to calculate a fitness factor for every insertion site based on its relative abundance in the inoculum and postinfection libraries. Eighty-nine transposon insertion mutants that were underrepresented after experimental infection in mice compared to their presence in the inocula were delineated as candidates for further evaluation. Genetically defined mutants lacking feoB (ferrous iron import), ddc (d-ala-d-ala-carboxypeptidase), and pntB (pyridine nucleotide transhydrogenase subunit) exhibited a fitness defect during systemic infection resulting from bacteremia. In vitro, these mutants, as well as a fepA (ferric enterobactin receptor) mutant, are defective in survival in human serum and within macrophages and are hypersensitive to killing by antimicrobial peptides compared to the survival of the parental strain under these conditions. Our data demonstrate that FepA is involved in the uptake of exogenous enterobactin in A. baumannii. Genetic complementation rescues the phenotypes of mutants in assays that emulate conditions encountered during infection. In summary, we have determined novel A. baumannii fitness genes involved in the pathogenesis of mammalian infection. IMPORTANCE A. baumannii is a significant cause of bacterial bloodstream infection in humans. Since multiple antibiotic resistance is becoming more common among strains of A. baumannii, there is an urgent need to develop novel tools to treat infections caused by this dangerous pathogen. To develop knowledge-guided treatment approaches for A. baumannii, a thorough understanding of the mechanism by which this pathogen causes bloodstream infection is required. Here, using a mouse model of infection, we report the identification of A. baumannii genes that are critical for the ability of this pathogen to cause bloodstream infections. This study lays the foundation for future research on A. baumannii genes that can be targeted to develop novel therapeutics against this emerging human pathogen.

ABSTRACT Klebsiella pneumoniae is an urgent public health threat because of resistance to carbapenems, antibiotics of last resort against Gram-negative bacterial infections. Despite the fact that K. pneumoniae is a leading cause of pneumonia in hospitalized patients, the bacterial factors required to cause disease are poorly understood. Insertion site sequencing combines transposon mutagenesis with high-throughput sequencing to simultaneously screen thousands of insertion mutants for fitness defects during infection. Using the recently sequenced K. pneumoniae strain KPPR1 in a well-established mouse model of pneumonia, insertion site sequencing was performed on a pool of >25,000 transposon mutants. The relative fitness requirement of each gene was ranked based on the ratio of lung to inoculum read counts and concordance between insertions in the same gene. This analysis revealed over 300 mutants with at least a 2-fold fitness defect and 69 with defects ranging from 10- to >2,000-fold. Construction of 6 isogenic mutants for use in competitive infections with the wild type confirmed their requirement for lung fitness. Critical fitness genes included those for the synthesis of branched-chain and aromatic amino acids that are essential in mice and humans, the transcriptional elongation factor RfaH, and the copper efflux pump CopA. The majority of fitness genes were conserved among reference strains representative of diverse pathotypes. These results indicate that regulation of outer membrane components and synthesis of amino acids that are essential to its host are critical for K. pneumoniae fitness in the lung. IMPORTANCE Klebsiella pneumoniae is a bacterium that commonly causes pneumonia in patients after they are admitted to the hospital. K. pneumoniae is becoming resistant to all available antibiotics, and when these infections spread to the bloodstream, over half of patients die. Since currently available antibiotics are failing, we must discover new ways to treat these infections. In this study, we asked what genes the bacterium needs to cause an infection, since the proteins encoded by these genes could be targets for new antibiotics. We identified over 300 genes that K. pneumoniae requires to grow in a mouse model of pneumonia. Many of the genes that we identified are found in K. pneumoniae isolates from throughout the world, including antibiotic-resistant forms. If new antibiotics could be made against the proteins that these genes encode, they may be broadly effective against K. pneumoniae.

ABSTRACT The urinary tract is one of the most common sites of infection in humans, and uropathogenic Escherichia coli (UPEC) is the main causative agent of urinary tract infections. Bacteria colonizing the urinary tract face extremely low iron availability. To counteract this, UPEC expresses a wide variety of iron acquisition systems. To exploit iron acquisition in UPEC as a global target for small-molecule inhibition, we developed and carried out a whole-cell growth-based high throughput screen of 149,243 compounds. Our primary assay was carried out under iron-limiting conditions. Hits in the primary screen were assayed using two counterscreens that ruled out iron chelators and compounds that inhibit growth by means other than inhibition of iron acquisition. We determined dose-response curves under two different iron conditions and purchased fresh compounds for selected hits. After retesting dose-response relationships, we identified 16 compounds that arrest growth of UPEC only under iron-limiting conditions. All compounds are bacteriostatic and do not inhibit proton motive force. A loss-of-target strategy was employed to identify the cellular target of these inhibitors. Two compounds lost inhibitory activity against a strain lacking TonB and were shown to inhibit irreversible adsorption of a TonB-dependent bacteriophage. Our results validate iron acquisition as a target for antibacterial strategies against UPEC and identify TonB as one of the cellular targets. IMPORTANCE Half of women will suffer at least one episode of urinary tract infection (UTI) during their lifetime. The current treatment for UTI involves antibiotic therapy. Resistance to currently used antibiotics has steadily increased over the last decade, generating a pressing need for the development of new therapeutic agents. Since iron is essential for colonization and scarce in the urinary tract, targeting iron acquisition would seem to be an attractive strategy. However, the multiplicity and redundancy of iron acquisition systems in uropathogenic Escherichia coli (UPEC) make it difficult to pinpoint a specific cellular target. Here, we identified 16 iron acquisition inhibitors through a whole-cell high-throughput screen, validating iron acquisition as a target for antibacterial strategies against UPEC. We also identified the cellular target of two of the inhibitors as the TonB system.

There is a critical gap in knowledge about how Gram-negative bacterial pathogens, using survival strategies developed for other niches, cause lethal bacteremia. Facultative anaerobic species of the Enterobacterales order are the most common cause of Gram-negative bacteremia, including Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, and Enterobacter hormaechei. Bacteremia often leads to sepsis, a life-threatening organ dysfunction resulting from unregulated immune responses to infection. Despite a lack of specialization for this host environment, Gram-negative pathogens cause nearly half of bacteremia cases annually. Based on our existing Tn-Seq fitness factor data from a murine model of bacteremia combined with comparative genomics of the five Enterobacterales species above, we prioritized 18 conserved fitness genes or operons for further characterization. Mutants were constructed for all genes in all five species. Each mutant was used to cochallenge C57BL/6 mice via tail vein injection along with each respective wild-type strain to determine competitive indices for each fitness gene. Five fitness factor genes, when mutated, attenuated mutants in four or five species in the spleen and liver (tatC, ruvA, gmhB, wzxE, arcA). Five additional fitness factor genes or operons were validated as outcompeted by wild-type in three, four, or five bacterial species in the spleen (xerC, prc, apaGH, atpG, aroC). Overall, 17 of 18 fitness factor mutants were attenuated in at least one species in the spleen or liver. Together, these findings allow for the development of a model of bacteremia pathogenesis that may include future targets of therapy against bloodstream infections.

ABSTRACT Proteus mirabilis rapidly migrates across surfaces using a periodic developmental process of differentiation alternating between short swimmer cells and elongated hyperflagellated swarmer cells. To undergo this vigorous flagellum-mediated motility, bacteria must generate a substantial proton gradient across their cytoplasmic membranes by using available energy pathways. We sought to identify the link between energy pathways and swarming differentiation by examining the behavior of defined central metabolism mutants. Mutations in the tricarboxylic acid (TCA) cycle (fumC and sdhB mutants) caused altered patterns of swarming periodicity, suggesting an aerobic pathway. Surprisingly, the wild-type strain swarmed on agar containing sodium azide, which poisons aerobic respiration; the fumC TCA cycle mutant, however, was unable to swarm on azide. To identify other contributing energy pathways, we screened transposon mutants for loss of swarming on sodium azide and found insertions in the following genes that involved fumarate metabolism or respiration: hybB, encoding hydrogenase; fumC, encoding fumarase; argH, encoding argininosuccinate lyase (generates fumarate); and a quinone hydroxylase gene. These findings validated the screen and suggested involvement of anaerobic electron transport chain components. Abnormal swarming periodicity of fumC and sdhB mutants was associated with the excretion of reduced acidic fermentation end products. Bacteria lacking SdhB were rescued to wild-type pH and periodicity by providing fumarate, independent of carbon source but dependent on oxygen, while fumC mutants were rescued by glycerol, independent of fumarate only under anaerobic conditions. These findings link multicellular swarming patterns with fumarate metabolism and membrane electron transport using a previously unappreciated configuration of both aerobic and anaerobic respiratory chain components. IMPORTANCE Bacterial locomotion and the existence of microbes were the first scientific observations that followed the invention of the microscope. A bacterium can swim through a fluid environment or coordinate motion with a group of bacteria and swarm across a surface. The flagellar motor, which propels the bacterium, is fueled by proton motive force. In contrast to the physiology that governs swimming motility, much less is known about the energy sources required for multicellular swarming on surfaces. In this study, we used Proteus mirabilis as a model organism to study vigorous swarming behavior and genetic and biochemical approaches to define energy pathways and central metabolism that contribute to multicellular motility. We found that swarming bacteria use a complete aerobic tricarboxylic acid (TCA) cycle but do not respire oxygen as the terminal electron acceptor, suggesting that multicellular cooperation during swarming reduces the amount of energy required by individual bacteria to achieve rapid motility.

Extraintestinal Escherichia coli (E. coli) evolved by acquisition of pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene transfer. Strains are heterogeneous but virulent uropathogenic isolates more often have specific fimbriae, toxins, and iron receptors than commensal strains. One may ask whether it is the virulence factors alone that are required to establish infection. While these virulence factors clearly contribute strongly to pathogenesis, bacteria must survive by metabolizing nutrients available to them. By constructing mutants in all major metabolic pathways and co-challenging mice transurethrally with each mutant and the wild type strain, we identified which major metabolic pathways are required to infect the urinary tract. We must also ask what else is E. coli doing in vivo? To answer this question, we examined the transcriptome of E. coli CFT073 in the murine model of urinary tract infection (UTI) as well as for E. coli strains collected and analyzed directly from the urine of patients attending either a urology clinic or a university health clinic for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene expression for these uropathogenic E. coli strains, identifying genes upregulated during murine and human UTI. Our findings allow us to propose a new definition of bacterial virulence.

ABSTRACT Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli. IMPORTANCE Uropathogenic Escherichia coli (UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in an E. coli K-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain, E. coli CFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged from E. coli K-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

ABSTRACT The increasing antibiotic resistance of pathogenic Escherichia coli species and the absence of a pan-protective vaccine pose major health concerns. We recently identified, by subtractive reverse vaccinology, nine Escherichia coli antigens that protect mice from sepsis. In this study, we characterized one of them, ECOK1_0290, named FdeC (factor adherence E. coli) for its ability to mediate E. coli adhesion to mammalian cells and extracellular matrix. This adhesive propensity was consistent with the X-ray structure of one of the FdeC domains that shows a striking structural homology to Yersinia pseudotuberculosis invasin and enteropathogenic E. coli intimin. Confocal imaging analysis revealed that expression of FdeC on the bacterial surface is triggered by interaction of E. coli with host cells. This phenotype was also observed in bladder tissue sections derived from mice infected with an extraintestinal strain. Indeed, we observed that FdeC contributes to colonization of the bladder and kidney, with the wild-type strain outcompeting the fdeC mutant in cochallenge experiments. Finally, intranasal mucosal immunization with recombinant FdeC significantly reduced kidney colonization in mice challenged transurethrally with uropathogenic E. coli, supporting a role for FdeC in urinary tract infections. IMPORTANCE Pathogenic Escherichia coli strains are involved in a diverse spectrum of diseases, including intestinal and extraintestinal infections (urinary tract infections and sepsis). The absence of a broadly protective vaccine against all these E. coli strains is a major problem for modern society due to high costs to health care systems. Here, we describe the structural and functional properties of a recently reported protective antigen, named FdeC, and elucidated its putative role during extraintestinal pathogenic E. coli infection by using both in vitro and in vivo infection models. The conservation of FdeC among strains of different E. coli pathotypes highlights its potential as a component of a broadly protective vaccine against extraintestinal and intestinal E. coli infections.

ABSTRACT The urinary tract is one of the most frequent sites of bacterial infection in humans. Uropathogenic Escherichia coli (UPEC) strains are the leading cause of urinary tract infections (UTIs) and are responsible for greater than 80% of uncomplicated cases in adults. Infection of the urinary tract occurs in an ascending manner, with colonization of the bladder leading to possible kidney infection and bacteremia. The goal of this study was to examine the population dynamics of UPEC in vivo using a murine model of ascending UTI. To track individual UPEC lineages within a host, we constructed 10 isogenic clones of UPEC strain CFT073 by inserting unique signature tag sequences between the pstS and glmS genes at the attTn7 chromosomal site. Mice were transurethrally inoculated with a mixture containing equal numbers of unique clones. After 4 and 48 h, the tags present in the bladders, kidneys, and spleens of infected mice were enumerated using tag-specific primers and quantitative real-time PCR. The results indicated that kidney infection and bacteremia associated with UTI are most likely the result of multiple rounds of ascension and dissemination from motile UPEC subpopulations, with a distinct bottleneck existing between the kidney and bloodstream. The abundance of tagged lineages became more variable as infection progressed, especially after bacterial ascension to the upper urinary tract. Analysis of the population kinetics of UPEC during UTI revealed metapopulation dynamics, with lineages that constantly increased and decreased in abundance as they migrated from one organ to another. IMPORTANCE Urinary tract infections are some of the most common infections affecting humans, and Escherichia coli is the primary cause in most uncomplicated cases. These infections occur in an ascending manner, with bacteria traveling from the bladder to the kidneys and potentially the bloodstream. Little is known about the spatiotemporal population dynamics of uropathogenic E. coli within a host. Here we describe a novel approach for tracking lineages of isogenic tagged E. coli strains within a murine host by the use of quantitative real-time PCR. Understanding the in vivo population dynamics and the factors that shape the bacterial population may prove to be of significant value in the development of novel vaccines and drug therapies.

Urinary tract infection (UTI) is the second most common infection in humans after those involving the respiratory tract. This results not only in huge annual economic costs, but in decreased workforce productivity and high patient morbidity. Most infections are caused by uropathogenic Escherichia coli (UPEC). Antibiotic treatment is generally effective for eradication of the infecting strain; however, documentation of increasing antibiotic resistance, allergic reaction to certain pharmaceuticals, alteration of normal gut flora, and failure to prevent recurrent infections represent significant barriers to treatment. As a result, approaches to prevent UTI such as vaccination represent a gap that must be addressed. Our laboratory has made progress toward development of a preventive vaccine against UPEC. The long-term research goal is to prevent UTIs in women with recurrent UTIs. Our objective has been to identify the optimal combination of protective antigens for inclusion in an effective UTI vaccine, optimal adjuvant, optimal dose, and optimal route of delivery. We hypothesized that a multi-subunit vaccine elicits antibody that protects against experimental challenge with UPEC strains. We have systematically identified four antigens that can individually protect experimentally infected mice from colonization of the bladder and/or kidneys by UPEC when administered intranasally with cholera toxin (CT) as an adjuvant. To advance the vaccine for utility in humans, we will group the individual antigens, all associated with iron acquisition (IreA, Hma, IutA, FyuA), into an effective combination to establish a multi-subunit vaccine. We demonstrated for all four vaccine antigens that antigen-specific serum IgG represents a strong correlate of protection in vaccinated mice. High antibody titers correlate with low colony forming units (CFUs) of UPEC following transurethral challenge of vaccinated mice. However, the contribution of cell-mediated immunity cannot be ruled out and must be investigated experimentally. We have demonstrated that antibodies bind to the surface of UPEC expressing the antigens. Sera from women with and without histories of UTI have been tested for antibody levels to vaccine antigens. Our results validate iron acquisition as a target for vaccination against UTI.

ABSTRACT Uropathogenic Escherichia coli (UPEC) strains, which cause the majority of uncomplicated urinary tract infections (UTIs), carry a unique assortment of virulence or fitness genes. However, no single defining set of virulence or fitness genes has been found in all strains of UPEC, making the differentiation between UPEC and fecal commensal strains of E. coli difficult without the use of animal models of infection or phylogenetic grouping. In the present study, we consider three broad categories of virulence factors simultaneously to better define a combination of virulence factors that predicts success in the urinary tract. A total of 314 strains of E. coli, representing isolates from fecal samples, asymptomatic bacteriuria, complicated UTIs, and uncomplicated bladder and kidney infections, were assessed by multiplex PCR for the presence of 15 virulence or fitness genes encoding adhesins, toxins, and iron acquisition systems. The results confirm previous reports of gene prevalence among isolates from different clinical settings and identify several new patterns of gene associations. One gene, tosA, a putative repeat-in-toxin (RTX) homolog, is present in 11% of fecal strains but 25% of urinary isolates. Whereas tosA-positive strains carry an unusually high number (11.2) of the 15 virulence or fitness genes, tosA-negative strains have an average of only 5.4 virulence or fitness genes. The presence of tosA was predictive of successful colonization of a murine model of infection, even among fecal isolates, and can be used as a marker of pathogenic strains of UPEC within a distinct subset of the B2 lineage. IMPORTANCE Escherichia coli is the primary cause of urinary tract infections, the most common bacterial infection of humans. Virulence of a uropathogenic strain is typically defined by the clinical source of the isolate, the ability to colonize the bladder and kidneys in a murine model, the phylogenetic group of the bacterium, and virulence gene content. Here we describe a novel single gene, the repeat-in-toxin gene tosA, the presence of which predicts virulence of E. coli isolates regardless of source. Rapid identification of uropathogenic strains of E. coli may aid in the development of therapeutic and preventive therapies.

ABSTRACT Infection with uropathogenic Escherichia coli (UPEC), the causative agent of most uncomplicated urinary tract infections, proceeds in an ascending manner and, if left untreated, may result in bacteremia and urosepsis. To examine the fate of UPEC after its entry into the bloodstream, we developed a murine model of sublethal bacteremia. CBA/J mice were inoculated intravenously with 1 × 106 CFU of pyelonephritis strain E. coli CFT073 carrying a bioluminescent reporter. Biophotonic imaging, used to monitor the infection over 48 h, demonstrated that the bacteria disseminated systemically and appeared to localize at discrete sites. UPEC was recovered from the spleen, liver, kidneys, lungs, heart, brain, and intestines as early as 20 min postinoculation, peaking at 24 h postinoculation. A nonpathogenic E. coli K-12 strain, however, disseminated at significantly lower levels (P < 0.01) and was cleared from the liver and cecum by 24 h postinoculation. Isogenic mutants lacking type 1 fimbriae, P fimbriae, capsule, TonB, the heme receptors Hma and ChuA, or particularly the sialic acid catabolism enzyme NanA were significantly outcompeted by wild-type CFT073 during bacteremia (P < 0.05), while flagellin and hemolysin mutants were not. IMPORTANCE E. coli is the primary cause of urinary tract infections. In severe cases of kidney infection, bacteria can enter the bloodstream and cause systemic disease. While the ability of E. coli to cause urinary tract infection has been extensively studied, the fate of these bacteria once they enter the bloodstream is largely unknown. Here we used an imaging technique to develop a mouse model of E. coli bloodstream infection and identify bacterial genes that are important for the bacteria to spread to and infect various organs. Understanding how urinary tract pathogens like E. coli cause disease after they enter the bloodstream may aid in the development of protective and therapeutic treatments.