Fitness factor genes conserved within the multi-species core genome of Gram-negative Enterobacterales species contribute to bacteremia pathogenesis.

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.

Klebsiella pneumoniae causes bacteremia using factors that mediate tissue-specific fitness and resistance to oxidative stress.

Gram-negative bacteremia is a major cause of global morbidity involving three phases of pathogenesis: initial site infection, dissemination, and survival in the blood and filtering organs. Klebsiella pneumoniae is a leading cause of bacteremia and pneumonia is often the initial infection. In the lung, K. pneumoniae relies on many factors like capsular polysaccharide and branched chain amino acid biosynthesis for virulence and fitness. However, mechanisms directly enabling bloodstream fitness are unclear. Here, we performed transposon insertion sequencing (TnSeq) in a tail-vein injection model of bacteremia and identified 58 K. pneumoniae bloodstream fitness genes. These factors are diverse and represent a variety of cellular processes. In vivo validation revealed tissue-specific mechanisms by which distinct factors support bacteremia. ArnD, involved in Lipid A modification, was required across blood filtering organs and supported resistance to soluble splenic factors. The purine biosynthesis enzyme PurD supported liver fitness in vivo and was required for replication in serum. PdxA, a member of the endogenous vitamin B6 biosynthesis pathway, optimized replication in serum and lung fitness. The stringent response regulator SspA was required for splenic fitness yet was dispensable in the liver. In a bacteremic pneumonia model that incorporates initial site infection and dissemination, splenic fitness defects were enhanced. ArnD, PurD, DsbA, SspA, and PdxA increased fitness across bacteremia phases and each demonstrated unique fitness dynamics within compartments in this model. SspA and PdxA enhanced K. pnuemoniae resistance to oxidative stress. SspA, but not PdxA, specifically resists oxidative stress produced by NADPH oxidase Nox2 in the lung, spleen, and liver, as it was a fitness factor in wild-type but not Nox2-deficient (Cybb-/-) mice. These results identify site-specific fitness factors that act during the progression of Gram-negative bacteremia. Defining K. pneumoniae fitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.

The Klebsiella pneumoniae ter Operon Enhances Stress Tolerance.

Healthcare-acquired infections are a leading cause of disease in patients that are hospitalized or in long-term-care facilities. Klebsiella pneumoniae (Kp) is a leading cause of bacteremia, pneumonia, and urinary tract infections in these settings. Previous studies have established that the ter operon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, the ter operon increases Kp fitness during gut colonization; however, the biologically relevant function of this operon is unknown. First, using a murine model of urinary tract infection, we demonstrate a novel role for the ter operon protein TerC as a bladder fitness factor. To further characterize TerC, we explored a variety of functions, including resistance to metal-induced stress, resistance to radical oxygen species-induced stress, and growth on specific sugars, all of which were independent of TerC. Then, using well-defined experimental guidelines, we determined that TerC is necessary for tolerance to ofloxacin, polymyxin B, and cetylpyridinium chloride. We used an ordered transposon library constructed in a Kp strain lacking the ter operon to identify the genes that are required to resist K2TeO3-induced and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3 disrupts the Kp cell envelope, though these effects are independent of ter. Collectively, the results from these studies indicate a novel role for the ter operon as a stress tolerance factor, thereby explaining its role in enhancing fitness in the gut and bladder.

A systematic analysis of hypermucoviscosity and capsule reveals distinct and overlapping genes that impact Klebsiella pneumoniae fitness.

Hypervirulent K. pneumoniae (hvKp) is a distinct pathotype that causes invasive community-acquired infections in healthy individuals. Hypermucoviscosity (hmv) is a major phenotype associated with hvKp characterized by copious capsule production and poor sedimentation. Dissecting the individual functions of CPS production and hmv in hvKp has been hindered by the conflation of these two properties. Although hmv requires capsular polysaccharide (CPS) biosynthesis, other cellular factors may also be required and some fitness phenotypes ascribed to CPS may be distinctly attributed to hmv. To address this challenge, we systematically identified genes that impact capsule and hmv. We generated a condensed, ordered transposon library in hypervirulent strain KPPR1, then evaluated the CPS production and hmv phenotypes of the 3,733 transposon mutants, representing 72% of all open reading frames in the genome. We employed forward and reverse genetic screens to evaluate effects of novel and known genes on CPS biosynthesis and hmv. These screens expand our understanding of core genes that coordinate CPS biosynthesis and hmv, as well as identify central metabolism genes that distinctly impact CPS biosynthesis or hmv, specifically those related to purine metabolism, pyruvate metabolism and the TCA cycle. Six representative mutants, with varying effect on CPS biosynthesis and hmv, were evaluated for their impact on CPS thickness, serum resistance, host cell association, and fitness in a murine model of disseminating pneumonia. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that hmv and CPS may serve distinct functions during pathogenesis. The integration of hmv and CPS to the metabolic status of the cell suggests that hvKp may require certain nutrients to specifically cause deep tissue infections.

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor.

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO3-2)-resistance (ter) operon as strongly associated with infection. However, TeO3-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.

Pathogenesis of Gram-Negative Bacteremia.

Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.

The ADP-Heptose Biosynthesis Enzyme GmhB is a Conserved Gram-Negative Bacteremia Fitness Factor.

Klebsiella pneumoniae is a leading cause of Gram-negative bacteremia, which is a major source of morbidity and mortality worldwide. Gram-negative bacteremia requires three major steps: primary site infection, dissemination to the blood, and bloodstream survival. Because K. pneumoniae is a leading cause of health care-associated pneumonia, the lung is a common primary infection site leading to secondary bacteremia. K. pneumoniae factors essential for lung fitness have been characterized, but those required for subsequent bloodstream infection are unclear. To identify K. pneumoniae genes associated with dissemination and bloodstream survival, we combined previously and newly analyzed insertion site sequencing (InSeq) data from a murine model of bacteremic pneumonia. This analysis revealed the gene gmhB as important for either dissemination from the lung or bloodstream survival. In Escherichia coli, GmhB is a partially redundant enzyme in the synthesis of ADP-heptose for the lipopolysaccharide (LPS) core. To characterize its function in K. pneumoniae, an isogenic knockout strain (ΔgmhB) and complemented mutant were generated. During pneumonia, GmhB did not contribute to lung fitness and did not alter normal immune responses. However, GmhB enhanced bloodstream survival in a manner independent of serum susceptibility, specifically conveying resistance to spleen-mediated killing. In a tail-vein injection of murine bacteremia, GmhB was also required by K. pneumoniae, E. coli, and Citrobacter freundii for optimal fitness in the spleen and liver. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance fitness in blood-filtering organs.

Management of Cytomegalovirus, Epstein-Barr Virus, and HIV Viral Load Quality Control Data Using Unity Real Time.

Quality control (QC) rules (Westgard rules) are applied to viral load testing to identify runs that should be reviewed or repeated, but this requires balancing the patient safety benefits of error detection with the cost and inefficiency of false rejection. In this study, we identified the total allowable errors (TEa) from the literature and utilized a commercially available software program (Unity Real Time; Bio-Rad Laboratories) to manage QC data, assess assay performance, and provide QC decision support for both FDA-approved/cleared (Abbott cytomegalovirus [CMV] and HIV viral load) as well as laboratory-developed (Epstein-Barr virus [EBV] viral load) assays. Unity Real Time was used to calculate means, standard deviations (SDs), and coefficient of variation (CV; in percent) of negative, low-positive, and high-positive control data from 73 to 83 days of testing. Sigma values were calculated to measure the test performance relative to a TEa of 0.5 log10. The sigma value of 5.06 for EBV predicts ∼230 erroneous results per million individual patient tests (0.02% frequency), whereas sigma values of >6 for CMV (11.32) and HIV (7.66) indicate <4 erroneous results per million individual patient tests. The Unity Real Time QC Design module utilized these sigma values to recommend QC rules and provided objective evidence for loosening the laboratory's existing QC rules for run acceptability, potentially reducing false rejection rates by 10-fold for the assay with the most variation (EBV viral load). This study provides a framework for laboratories, with Unity Real Time as a tool, to evaluate assay performance relative to clinical decision points and establish optimal rules for routine monitoring of molecular viral load assay performance.

Proceedings of the Clinical Microbiology Open 2018 and 2019 - a Discussion about Emerging Trends, Challenges, and the Future of Clinical Microbiology.

Clinical Microbiology Open (CMO), a meeting supported by the American Society for Microbiology's Clinical and Public Health Microbiology Committee (CPHMC) and Corporate Council, provides a unique interactive platform for leaders from diagnostic microbiology laboratories, industry, and federal agencies to discuss the current and future state of the clinical microbiology laboratory. The purpose is to leverage the group's diverse views and expertise to address critical challenges, and discuss potential collaborative opportunities for diagnostic microbiology, through the utilization of varied resources. The first and second CMO meetings were held in 2018 and 2019, respectively. Discussions were focused on the diagnostic potential of innovative technologies and laboratory diagnostic stewardship, including expansion of next-generation sequencing into clinical diagnostics, improvement and advancement of molecular diagnostics, emerging diagnostics, including rapid antimicrobial susceptibility and point of care testing (POCT), harnessing big data through artificial intelligence, and staffing in the clinical microbiology laboratory. Shortly after CMO 2019, the coronavirus disease 2019 (COVID-19) pandemic further highlighted the need for the diagnostic microbiology community to work together to utilize and expand on resources to respond to the pandemic. The issues, challenges, and potential collaborative efforts discussed during the past two CMO meetings proved critical in addressing the COVID-19 response by diagnostic laboratories, industry partners, and federal organizations. Planning for a third CMO (CMO 2022) is underway and will transition from a discussion-based meeting to an action-based meeting. The primary focus will be to reflect on the lessons learned from the COVID-19 pandemic and better prepare for future pandemics.

Prolonged Severe Acute Respiratory Syndrome Coronavirus 2 Replication in an Immunocompromised Patient.

We describe a case of chronic coronavirus disease 2019 (COVID-19) in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for at least 119 days. The patient had 3 admissions related to COVID-19 over a 4-month period and was treated twice with remdesivir and convalescent plasma with resolution of symptoms. The patient's lack of seroconversion and prolonged course illustrate the importance of humoral immunity in resolving SARS-CoV-2 infection. This case highlights challenges in managing immunocompromised hosts, who may act as persistent shedders and sources of transmission.

The Klebsiella pneumoniae citrate synthase gene, gltA, influences site specific fitness during infection.

Klebsiella pneumoniae (Kp), one of the most common causes of healthcare-associated infections, increases patient morbidity, mortality, and hospitalization costs. Kp must acquire nutrients from the host for successful infection; however, the host is able to prevent bacterial nutrient acquisition through multiple systems. This includes the innate immune protein lipocalin 2 (Lcn2), which prevents Kp iron acquisition. To identify novel Lcn2-dependent Kp factors that mediate evasion of nutritional immunity during lung infection, we undertook an InSeq study using a pool of >20,000 transposon mutants administered to Lcn2+/+ and Lcn2-/- mice. Comparing transposon mutant frequencies between mouse genotypes, we identified the Kp citrate synthase, GltA, as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly, in vitro studies suggest that this interaction is not direct. Given that GltA is involved in oxidative metabolism, we screened the ability of this mutant to use a variety of carbon and nitrogen sources. The results indicated that the gltA mutant has a distinct amino acid auxotrophy rendering it reliant upon glutamate family amino acids for growth. Deletion of Lcn2 from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, corresponding to increased fitness of the gltA mutant in vivo and ex vivo. Accordingly, addition of glutamate family amino acids to Lcn2+/+ bronchioloalveolar lavage fluid rescued growth of the gltA mutant. Using a variety of mouse models of infection, we show that GltA is an organ-specific fitness factor required for complete fitness in the spleen, liver, and gut, but dispensable in the bloodstream. Similar to bronchioloalveolar lavage fluid, addition of glutamate family amino acids to Lcn2+/+ organ lysates was sufficient to rescue the loss of gltA. Together, this study describes a critical role for GltA in Kp infection and provides unique insight into how metabolic flexibility impacts bacterial fitness during infection.

Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection.

The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality.

TnseqDiff: identification of conditionally essential genes in transposon sequencing studies.

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.

Bacterial siderophores that evade or overwhelm lipocalin 2 induce hypoxia inducible factor 1α and proinflammatory cytokine secretion in cultured respiratory epithelial cells.

Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.

Gut community structure as a risk factor for infection in Klebsiella pneumoniae-colonized patients.

The primary risk factor for infection with members of the Klebsiella pneumoniae species complex is prior gut colonization, and infection is often caused by the colonizing strain. Despite the importance of the gut as a reservoir for infectious K. pneumoniae, little is known about the association between the gut microbiome and infection. To explore this relationship, we undertook a case-control study comparing the gut community structure of K. pneumoniae-colonized intensive care and hematology/oncology patients. Cases were K. pneumoniae-colonized patients infected by their colonizing strain (N = 83). Controls were K. pneumoniae-colonized patients who remained asymptomatic (N = 149). First, we characterized the gut community structure of K. pneumoniae-colonized patients agnostic to case status. Next, we determined that gut community data is useful for classifying cases and controls using machine learning models and that the gut community structure differed between cases and controls. K. pneumoniae relative abundance, a known risk factor for infection, had the greatest feature importance, but other gut microbes were also informative. Finally, we show that integration of gut community structure with bacterial genotype data enhanced the ability of machine learning models to discriminate cases and controls. Interestingly, inclusion of patient clinical variables failed to improve the ability of machine learning models to discriminate cases and controls. This study demonstrates that including gut community data with K. pneumoniae-derived biomarkers improves our ability to classify infection in K. pneumoniae-colonized patients.IMPORTANCEColonization is generally the first step in pathogenesis for bacteria with pathogenic potential. This step provides a unique window for intervention since a given potential pathogen has yet to cause damage to its host. Moreover, intervention during the colonization stage may help alleviate the burden of therapy failure as antimicrobial resistance rises. Yet, to understand the therapeutic potential of interventions that target colonization, we must first understand the biology of colonization and if biomarkers at the colonization stage can be used to stratify infection risk. The bacterial genus Klebsiella includes many species with varying degrees of pathogenic potential. Members of the K. pneumoniae species complex have the highest pathogenic potential. Patients colonized in their gut by these bacteria are at higher risk of subsequent infection with their colonizing strain. However, we do not understand if other members of the gut microbiota can be used as a biomarker to predict infection risk. In this study, we show that the gut microbiota differs between colonized patients who develop an infection versus those who do not. Additionally, we show that integrating gut microbiota data with bacterial factors improves the ability to classify infections. Surprisingly, patient clinical factors were not useful for classifying infections alone or when added to microbiota-based models. This indicates that the bacterial genotype and the microbial community in which it exists may determine the progression to infection. As we continue to explore colonization as an intervention point to prevent infections in individuals colonized by potential pathogens, we must develop effective means for predicting and stratifying infection risk.

Bloodstream infections: mechanisms of pathogenesis and opportunities for intervention.

Bloodstream infections (BSIs) are common in hospitals, often life-threatening and increasing in prevalence. Microorganisms in the blood are usually rapidly cleared by the immune system and filtering organs but, in some cases, they can cause an acute infection and trigger sepsis, a systemic response to infection that leads to circulatory collapse, multiorgan dysfunction and death. Most BSIs are caused by bacteria, although fungi also contribute to a substantial portion of cases. Escherichia coli, Staphylococcus aureus, coagulase-negative Staphylococcus, Klebsiella pneumoniae and Candida albicans are leading causes of BSIs, although their prevalence depends on patient demographics and geographical region. Each species is equipped with unique factors that aid in the colonization of initial sites and dissemination and survival in the blood, and these factors represent potential opportunities for interventions. As many pathogens become increasingly resistant to antimicrobials, new approaches to diagnose and treat BSIs at all stages of infection are urgently needed. In this Review, we explore the prevalence of major BSI pathogens, prominent mechanisms of BSI pathogenesis, opportunities for prevention and diagnosis, and treatment options.

Infection characteristics among Serratia marcescens capsule lineages.

Serratia marcescens is a healthcare-associated pathogen that causes bloodstream infections, pneumonia, and urinary tract infections. The capsule polysaccharide of S. marcescens is a critical fitness determinant during infection and recent work established the relationship between capsule locus (KL) genetic sequences within the species. Strains belonging to KL1 and KL2 capsule clades produce sialylated polysaccharides and represent the largest subpopulation of isolates from clinical origin while the S. marcescens type strain and other environmental isolates were classified as KL5. In this work, the contribution of these and other capsules to pathogenesis in multiple infection models was determined. Using a murine tail vein injection model of bacteremia, clinical strains demonstrated capsule-dependent colonization of spleen, liver, and kidney following inoculation. The KL5 strain, in contrast, exhibited no loss of survival in this model when capsule genes were deleted. Furthermore, the wild-type KL5 strain was cleared more rapidly from both the spleen and liver compared to a KL1 strain. Similar results were observed in a bacteremic pneumonia model in that all tested strains of clinical origin demonstrated a requirement for capsule in both the primary lung infection site and for bloodstream dissemination to other organs. Finally, strains from each KL clade were tested for the role of capsule in internalization by bone marrow-derived macrophages. Only the sialylated KL1 and KL2 clade strains, representing the majority of clinical isolates, exhibited capsule-dependent inhibition of internalization, suggesting that capsule-mediated resistance to macrophage phagocytosis may enhance survival and antibacterial defenses during infection.

Gut community structure as a risk factor for infection in Klebsiella -colonized patients.

The primary risk factor for infection with members of the Klebsiella pneumoniae species complex is prior gut colonization, and infection is often caused by the colonizing strain. Despite the importance of the gut as a reservoir for infectious Klebsiella , little is known about the association between the gut microbiome and infection. To explore this relationship, we undertook a case-control study comparing the gut community structure of Klebsiella -colonized intensive care and hematology/oncology patients. Cases were Klebsiella -colonized patients infected by their colonizing strain (N = 83). Controls were Klebsiella -colonized patients that remained asymptomatic (N = 149). First, we characterized the gut community structure of Klebsiella -colonized patients agnostic to case status. Next, we determined that gut community data is useful for classifying cases and controls using machine learning models and that the gut community structure differed between cases and controls. Klebsiella relative abundance, a known risk factor for infection, had the greatest feature importance but other gut microbes were also informative. Finally, we show that integration of gut community structure with bacterial genotype or clinical variable data enhanced the ability of machine learning models to discriminate cases and controls. This study demonstrates that including gut community data with patient- and Klebsiella -derived biomarkers improves our ability to predict infection in Klebsiella -colonized patients. Importance: Colonization is generally the first step in pathogenesis for bacteria with pathogenic potential. This step provides a unique window for intervention since a given potential pathogen has yet to cause damage to its host. Moreover, intervention during the colonization stage may help alleviate the burden of therapy failure as antimicrobial resistance rises. Yet, to understand the therapeutic potential of interventions that target colonization, we must first understand the biology of colonization and if biomarkers at the colonization stage can be used to stratify infection risk. The bacterial genus Klebsiella includes many species with varying degrees of pathogenic potential. Members of the K. pneumoniae species complex have the highest pathogenic potential. Patients colonized in their gut by these bacteria are at higher risk of subsequent infection with their colonizing strain. However, we do not understand if other members of the gut microbiota can be used as a biomarker to predict infection risk. In this study, we show that the gut microbiota differs between colonized patients that develop an infection versus those that do not. Additionally, we show that integrating gut microbiota data with patient and bacterial factors improves the ability to predict infections. As we continue to explore colonization as an intervention point to prevent infections in individuals colonized by potential pathogens, we must develop effective means for predicting and stratifying infection risk.

Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia.

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.

The ArcAB Two-Component System: Function in Metabolism, Redox Control, and Infection.

ArcAB, also known as the Arc system, is a member of the two-component system family of bacterial transcriptional regulators and is composed of sensor kinase ArcB and response regulator ArcA. In this review, we describe the structure and function of these proteins and assess the state of the literature regarding ArcAB as a sensor of oxygen consumption. The bacterial quinone pool is the primary modulator of ArcAB activity, but questions remain for how this regulation occurs. This review highlights the role of quinones and their oxidation state in activating and deactivating ArcB and compares competing models of the regulatory mechanism. The cellular processes linked to ArcAB regulation of central metabolic pathways and potential interactions of the Arc system with other regulatory systems are also reviewed. Recent evidence for the function of ArcAB under aerobic conditions is challenging the long-standing characterization of this system as strictly an anaerobic global regulator, and the support for additional ArcAB functionality in this context is explored. Lastly, ArcAB-controlled cellular processes with relevance to infection are assessed.