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The primary risk factor for infection with members ofthe Klebsiella pneumoniaespecies complex is prior gut colonization, and infection is often caused by the colonizing strain. Despite the importance of the gut as a reservoir for infectiousKlebsiella, 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 ofKlebsiella-colonized intensive care and hematology/oncology patients. Cases wereKlebsiella-colonized patients infected by their colonizing strain (N = 83). Controls wereKlebsiella-colonized patients that remained asymptomatic (N = 149). First, we characterized the gut community structure ofKlebsiella-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.Klebsiellarelative 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- andKlebsiella-derived biomarkers improves our ability to predict infection inKlebsiella-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 genusKlebsiellaincludes many species with varying degrees of pathogenic potential. Members of theK. pneumoniaespecies 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.

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 pneumoniaeis a leading cause of bacteremia and pneumonia is often the initial infection. In the lung,K. pneumoniaerelies 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 58K. pneumoniaebloodstream fitness genes. These factors are diverse and represent a variety of cellular processes.In vivovalidation 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 largely enhanced liver fitness 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, and DsbA, SspA, and PdxA increased fitness across bacteremia phases. SspA and PdxA enhancedK. pnuemoniaeresistance to oxidative stress. SspA 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. DefiningK. pneumoniaefitness strategies across bacteremia phases could illuminate therapeutic targets that prevent infection and sepsis.Author SummaryGram-negative bacteremia is a deadly family of infections that initiate sepsis, a leading cause of global morbidity and mortality. Only a small number of Gram-negative species contribute to the majority of clinical bacteremia.Klebsiella pneumoniaeis the second leading cause of Gram-negative bacteremia, and the third leading cause of overall bloodstream infection.K. pneumoniaeis highly linked to hospital-associated infection with increasing antimicrobial resistance, endangering the most vulnerable patients. It is critical to understand the pathogenesis ofK. pneumoniaebacteremia to better develop targets for future therapies that can prevent these deadly infections. Here, we define over 50K. pneumoniaegenes that support bloodstream fitness. These factors are diverse, support tissue-specific fitness, and increase bacterial resistance to oxidative stress. Our study is the first to systematically defineK. pneumoniaefactors enhancing bacteremia in a mammalian system. These results illuminate host-pathogen interactions duringK. pneumoniaebacteremia that may be extended to additional Gram-negative species.

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 (K 2 TeO 3 ) resistance, is associated with infection in colonized patients.

Despite EMS-implemented screening and treatment protocols for suspected sepsis patients, prehospital fluid therapy is variable. We sought to describe prehospital fluid administration in suspected sepsis patients, including demographic and clinical factors associated with fluid outcomes. A retrospective cohort of adult patients from a large, county-wide EMS system from January 2018-February 2020 was identified. Patient care reports for suspected sepsis were included, as identified by EMS clinician impression of sepsis, or keywords “sepsis” or “septic” in the narrative. Outcomes were the proportions of suspected sepsis patients for whom intravenous (IV) therapy was attempted and those who received ≥500 mL IV fluid if IV access was successful. Associations between patient demographics and clinical factors with fluid outcomes were estimated with multivariable logistic regression adjusting for transport interval. Of 4,082 suspected sepsis patients identified, the mean patient age was 72.5 (SD 16.2) years, 50.6% were female, and 23.8% were Black. Median (interquartile range [IQR]) transport interval was 16.5 (10.9–23.2) minutes. Of identified patients, 1,920 (47.0%) had IV fluid therapy attempted, and IV access was successful in 1,872 (45.9%). Of those with IV access, 1,061 (56.7%) received ≥500mL of fluid from EMS. In adjusted analyses, female (versus male) sex (odds ratio [OR] 0.79, 95% confidence interval [CI] 0.69–0.90), Black (versus White) race (OR 0.57, 95% CI 0.49–0.68), and end stage renal disease (OR 0.51, 95% CI 0.32–0.82) were negatively associated with attempted IV therapy. Systolic blood pressure (SBP) 20 (OR 1.90, 95% CI 1.61–2.23) were positively associated with attempted IV therapy. Female sex (OR 0.72, 95% CI 0.59–0.88) and congestive heart failure (CHF) (OR 0.55, 95% CI 0.40–0.75) were negatively associated with receiving goal fluid volume while SBP 100.4 F or

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 theteroperon, a genetic locus that confers tellurite oxide (K2TeO3) resistance, is associated with infection in colonized patients. Rather than enhancing fitness during infection, theteroperon 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 theteroperon 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 ROS-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 theteroperon to identify genes required to resist K2TeO3− and polymyxin B-induced stress, which suggested that K2TeO3-induced stress is experienced at the bacterial cell envelope. Finally, we confirmed that K2TeO3disrupts the Kp cell envelope, though these effects are independent ofter. Collectively, the results from these studies indicate a novel role for theteroperon as stress tolerance factor, therefore explaining its role in enhancing fitness in the gut and bladder.

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.

Background Diagnostic sensitivities of point-of-care SARS-CoV-2 assays depend on specimen type and population-specific viral loads. Evaluation of these assays require “direct” specimens from paired-swab studies rather than more accessible residual specimens in viral transport media (VTM). Methods Residual VTM and limit-of-detection studies were conducted on Abbott ID NOW™ COVID-19, Quidel Sofia 2™ SARS Antigen FIA, and DiaSorin Simplexa™ COVID-19 Direct assays, with cycle threshold (CT) adjustments to approximate direct-specimen testing based on gene-target doubling each PCR cycle. Logistic regression was used to model assay performance by specimen CT. These models were applied to CT distributions of symptomatic and asymptomatic populations presenting to emergency services to predict the percentage of specimens that would be detected by each assay. A 96-sample paired-swab study was conducted to confirm model results. Results When using direct nasopharyngeal samples and fit with either VTM or limit-of-detection data, percent positivities for ID NOW (symptomatic 94.9%/97.4%; asymptomatic 88.4.0%/89.6%) and Simplexa (symptomatic 97.8%/97.2%; asymptomatic 91.1%/90.8%) were predicted to be similar. Likewise, percent positivities for ID NOW with direct nasal specimens (symptomatic 77.8%; asymptomatic 64.5%) and, fit with VTM data, Sofia 2 with direct nasopharyngeal specimens (symptomatic 76.6%, asymptomatic 60.3%) were similar. The paired-swab study comparing direct nasopharyngeal specimens on ID NOW and nasopharyngeal VTM specimens on Simplexa showed 99% concordance. Conclusions Assay performance can be modeled as dependent on viral load, fit using laboratory bench study results, and adjusted to account for direct-specimen testing. When using nasopharyngeal specimens, direct testing on Abbott ID NOW and VTM testing on DiaSorin Simplexa have similar performance.

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. Since K. pneumoniae is a leading cause of healthcare-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 performed insertion site sequencing (InSeq) using a pool of >25,000 transposon mutants in 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 bloodstream survival. Together, this study identifies GmhB as a conserved Gram-negative bacteremia fitness factor that acts through LPS-mediated mechanisms to enhance bloodstream survival.IMPORTANCEKlebsiella pneumoniae frequently causes healthcare-associated infections including pneumonia and bacteremia. This is particularly concerning due to emerging antimicrobial resistance and the propensity for bacteremia to initiate sepsis, which has high mortality and is the most expensive hospital-treated condition. Defining mechanisms of bloodstream survival is critical to understanding the pathology of bacteremia and identifying novel targets for future therapies. In this study, we identified the K. pneumoniae enzyme GmhB as a bloodstream-specific fitness factor that enables the bacteria to survive in the spleen but is dispensable in the lung. Furthermore, GmhB is also needed by the related bacterial pathogens Escherichia coli and Citrobacter freundii to cause bacteremia. Conserved bacteremia fitness factors such a GmhB could be the basis for future therapeutics that would alleviate significant disease caused by from multiple diverse pathogens.

The likelihood of survival from ventricular fibrillation (VF) declines 7%-10% per minute until successful defibrillation. When VF duration is prolonged, immediate defibrillation of the ischemic myocardium is less likely to result in ROSC, and repeated unsuccessful defibrillations are associated with post-resuscitation myocardial dysfunction. Thus, the timing of defibrillation should be based upon the probability of shock success-a function of VF duration. Unfortunately, VF duration is often unknown in out-of-hospital cardiac arrest (OHCA) and a better predictor of shock success is needed.To assess the ability of end-tidal carbon dioxide (EtCOThis retrospective study included adult patients among four EMS systems who experienced non-traumatic OHCA from August, 2015-July, 2017 and received one or more defibrillations. First and succedent shocks were analyzed separately. First shocks represented EMS-attempted defibrillation of patients who had not received a prior AED shock, whereas succedent shocks included all shocks subsequent to the first. Logistic regression provided odds ratios (OR) for first shocks resulting in ROSC, while a generalized estimating equation was used to analyze succedent shocks.Among 324 patients, 869 shocks were delivered by EMS (153 first and 716 succedent shocks). Layperson CPR was performed in 48.1% of cases and 21.6% received an AED shock before EMS arrival. First defibrillation ROSC was more likely with layperson CPR (OR = 4.41;p = 0.01) and increasing EtCOAn optimal defibrillation threshold EtCO2 of 27 and 32 mmHg was observed for patients with and without layperson CPR, respectively. Further studies are warranted to verify these results and to evaluate the clinical effect of delaying defibrillation in favor of chest compressions until these values are attained.

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 CMV and HIV viral load) as well as laboratory-developed (EBV viral load) assays. Unity Real Time was used to calculate means, SDs, and %CV of negative, low positive and high positive control data from 73-83 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 >6 for CMV (11.32) and HIV (7.66) indicate

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

Klebsiella pneumoniae and the closely related species K. variicola and K. quasipneumoniae are common causes of health care-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed. A multiplex quantitative PCR (qPCR) assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and a preceding or coincident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. qPCR from rectal swab samples was used to measure the association between the relative abundance of Klebsiella and subsequent infections. The Klebsiella relative abundance by qPCR was highly correlated with 16S sequencing (ρ = 0.79; P < 0.001). The median Klebsiella relative abundance was higher in cases (15.7% [interquartile range {IQR}, 0.93 to 52.6%]) (n = 83) than in controls (1.01% [IQR, 0.02 to 12.8%]) (n = 155) (P < 0.0001). Adjusting for multiple clinical covariates using inverse probability of treatment weighting, a Klebsiella relative abundance of >22% was associated with infection overall (odds ratio [OR], 2.87 [95% confidence interval {CI}, 1.64 to 5.03]) (P = 0.0003) and with bacteremia in a secondary analysis (OR, 4.137 [95% CI, 1.448 to 11.818]) (P = 0.0084). Measurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection. IMPORTANCE Colonization by bacterial pathogens often precedes infection and offers a window of opportunity to prevent these infections in the first place. Klebsiella colonization is significantly and reproducibly associated with subsequent infection; however, factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, a high Klebsiella density, defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available PCR technology, this type of assay could be deployed in clinical laboratories to identify patients at an increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention intervention.

Klebsiella commonly colonizes the intestinal tract of hospitalized patients and is a leading cause of health care-associated infections. Colonization is associated with subsequent infection, but the factors determining this progression are unclear. A cohort study was performed, in which intensive care and hematology/oncology patients with Klebsiella colonization based on rectal swab culture were enrolled and monitored for infection for 90 days after a positive swab. Electronic medical records were analyzed for patient factors associated with subsequent infection, and variables of potential significance in a bivariable analysis were used to build a final multivariable model. Concordance between colonizing and infecting isolates was assessed by wzi capsular gene sequencing. Among 2,087 hospitalizations from 1,978 colonized patients, 90 cases of infection (4.3%) were identified. The mean time to infection was 20.6 ± 24.69 (range, 0 to 91; median, 11.5) days. Of 86 typed cases, 68 unique wzi types were identified, and 69 cases (80.2%) were colonized with an isolate of the same type prior to infection. Based on multivariable modeling, overall comorbidities, depression, and low albumin levels at the time of rectal swab collection were independently associated with subsequent Klebsiella infection (i.e., cases). Despite the high diversity of colonizing strains of Klebsiella, there is high concordance with subsequent infecting isolates, and progression to infection is relatively quick. Readily accessible data from the medical record could be used by clinicians to identify colonized patients at an increased risk of subsequent Klebsiella infection. IMPORTANCE Klebsiella is a leading cause of health care-associated infections. Patients who are intestinally colonized with Klebsiella are at a significantly increased risk of subsequent infection, but only a subset of colonized patients progress to disease. Colonization offers a potential window of opportunity to intervene and prevent these infections, if the patients at greatest risk could be identified. To identify patient factors associated with infection in colonized patients, we studied 1,978 colonized patients. We found that patients with a higher burden of underlying disease in general, depression in particular, and low albumin levels in a blood test were more likely to develop infection. However, these variables did not completely predict infection, suggesting that other host and microbial factors may also be important. The clinical variables associated with infection are readily available in the medical record and could serve as the foundation for developing an integrated risk assessment of Klebsiella infection in hospitalized patients.

Clinical Enterobacteriacae isolates with a colistin minimum inhibitory concentration (MIC) ≥4 mg/L from a United States hospital were screened for the mcr-1 gene using real-time polymerase chain reaction (RT-PCR) and confirmed by whole-genome sequencing. Four colistin-resistant Escherichia coli isolates contained mcr-1. Two isolates belonged to the same sequence type (ST-632). All subjects had prior international travel and antimicrobial exposure.

Background Despite the recent emergence of plasmid-mediated colistin resistance, the epidemiology and mechanisms of colistin-resistant Enterobacterales (CORE) infections remain poorly understood. Methods A case–case–control study was conducted utilizing routine clinical isolates obtained at a single tertiary health system in Ann Arbor, Michigan. Patients with CORE isolates from January 1, 2016, to March 31, 2017, were matched 1:1 with patients with colistin-susceptible Enterobacterales (COSE) and uninfected controls. Multivariable logistic regression was used to compare clinical and microbiologic features of patients with CORE and COSE to controls. A subset of available CORE isolates underwent whole-genome sequencing to identify putative colistin resistance genes. Results Of 16 373 tested clinical isolates, 166 (0.99%) were colistin-resistant, representing 103 unique patients. Among 103 CORE isolates, 103 COSE isolates, and 102 uninfected controls, antibiotic exposure in the antecedent 90 days and age >55 years were predictors of both CORE and COSE. Of 33 isolates that underwent whole-genome sequencing, a large variety of mutations associated with colistin resistance were identified, including 4 mcr-1/mcr-1.1 genes and 4 pmrA/B mutations among 9 Escherichia coli isolates and 5 mgrB and 3 PmrA mutations among 8 Klebsiella pneumoniae isolates. Genetic mutations found in Enterobacter species were not associated with known phenotypic colistin resistance. Conclusions Increased age and prior antibiotic receipt were associated with increased risk for patients with CORE and for patients with COSE. Mcr-1, pmrA/B, and mgrB were the predominant colistin resistance–associated mutations identified among E. coli and K. pneumoniae, respectively. Mechanisms of colistin resistance among Enterobacter species could not be determined.

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., Author summary The bacterial pathogen Klebsiella pneumoniae is of substantial public health concern due to its ability to cause serious antibiotic-resistant infections. These infections frequently occur in healthcare settings, especially in patients with detectable gut colonization by K. pneumoniae. Importantly, infectious K. pneumoniae strains are often detected in the gut of patients with K. pneumoniae disease, indicating that the gut is a reservoir of infectious K. pneumoniae. Our previous work interrogating the genetic underpinnings of K. pneumoniae disease in colonized patients identified a strong association between K. pneumoniae infection and the presence of an enigmatic genetic locus known as the ter operon. We found that this operon is not needed for pneumonia and bacteremia, and therefore, we explored the importance of the ter operon in the gut. K. pneumoniae lacking ter function was at a disadvantage in the gut, thus explaining the connection between the ter operon and infection in hospitalized patients. Interestingly, the advantage conferred by the ter operon in the gut was associated with the presence of specific indigenous gut microbiota and the presence of short-chain fatty acids, which are metabolized by the host and gut microbiota. This work demonstrates that the ter operon is a microbiome-dependent gut fitness factor and suggests that indigenous gut bacteria may limit colonization by infectious K. pneumoniae.

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.

BackgroundKlebsiella commonly colonizes the intestinal tract of hospitalized patients and is a leading cause of healthcare-associated infections. Colonization is associated with subsequent infection, but the factors determining this progression are unclear.MethodsIntensive care and hematology/oncology patients were screened for Klebsiella colonization by rectal swab culture and monitored for infection for 90 days after a positive swab. Electronic medical records were analyzed for patient factors associated with subsequent infection, and variables of potential significance in bivariable analysis were used to build a final multivariable model. Concordance between colonizing and infecting isolates was assessed by wzi capsular gene sequencing.ResultsAmong 2087 hospital encounters from 1978 colonized patients, 90 cases of infection (4.3%) were identified. Mean time to infection was 20.6 ±24.69 (range 0-91, median 11.5) days. Of 86 typed cases, 68 unique wzi types were identified and 69 cases (80.2%) were colonized with an isolate of the same type prior to infection. Based on multivariable modeling, overall comorbidities, depression and low albumin level at the time of rectal swab were independently associated with subsequent Klebsiella infection.ConclusionsDespite the high diversity of colonizing strains of Klebsiella, there is high concordance with subsequent infecting isolates and progression to infection is relatively quick. Readily accessible data from the medical record could be used by clinicians to identify colonized patients at increased risk of subsequent Klebsiella infection.ImportanceKlebsiella is a leading cause of healthcare-associated infections. Patients who are intestinally colonized with Klebsiella are at significantly increased risk of subsequent infection, but only a subset of colonized patients progress to disease. Colonization offers a potential window of opportunity to intervene and prevent these infections, if the patients at greatest risk could be identified. To identify patient factors associated with infection in colonized patients, we studied 1978 colonized patients. We found that patients with a higher burden of underlying disease in general, depression in particular, and low albumin in a blood test were more likely to be a case of infection. However, these variables did not completely predict infection, suggesting that other host and microbial factors may also be important. The average time to infection was 3 weeks, suggesting that there is time to intervene and prevent Klebsiella infections in hospitalized patients.

BackgroundKlebsiella pneumoniae and closely related species K. variicola and K. quasipneumoniae are common causes of healthcare-associated infections, and patients frequently become infected with their intestinal colonizing strain. To assess the association between Klebsiella colonization density and subsequent infections, a case-control study was performed.MethodsA multiplex qPCR assay was developed and validated to quantify Klebsiella (K. pneumoniae, K. variicola, and K. quasipneumoniae combined) relative to total bacterial DNA copies in rectal swabs. Cases of Klebsiella infection were identified based on clinical definitions and having a clinical culture isolate and preceding or co-incident colonization isolate with the same wzi capsular sequence type. Controls were colonized patients without subsequent infection and were matched 2:1 to cases based on age, sex, and rectal swab collection date. Quantitative PCR (qPCR) from rectal swab samples was used to measure the association between relative abundance (RA) of Klebsiella and subsequent infections.ResultsKlebsiella RA by qPCR highly correlated with 16S sequencing (ρ=0.79; P Klebsiella RA in the study group was 2.6% (interquartile range (IQR) 0.1-22.5, n=238), and was higher in cases (15.7%, IQR 0.93-52.6%, n=83) than controls (1.01%, IQR 0.02-12.8%; n=155; P Klebsiella RA > 22% had a 2.87-fold (1.64-5.03, P =0.0003) increased odds of infection compared to those with lower colonization density levels.ConclusionsMeasurement of colonization density by qPCR could represent a novel approach to identify hospitalized patients at risk for Klebsiella infection.ImportanceColonization by bacterial pathogens often precedes infection, and offers a window of opportunity to prevent these infections. Klebsiella colonization is significantly and reproducibly associated with subsequent infection, however factors that enhance or mitigate this risk in individual patients are unclear. This study developed an assay to measure the density of Klebsiella colonization, relative to total fecal bacteria, in rectal swabs from hospitalized patients. Applying this assay to 238 colonized patients, high Klebsiella density defined as >22% of total bacteria, was significantly associated with subsequent infection. Based on widely available polymerase chain reaction (PCR) technology, this type of assay could be deployed in clinical laboratories to identify patients at increased risk of Klebsiella infections. As novel therapeutics are developed to eliminate pathogens from the gut microbiome, a rapid Klebsiella colonization density assay could identify patients who would benefit from this type of infection prevention interventions.

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., Author summary Klebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

BackgroundEpidemiologic studies have linked antibiotic exposure to subsequent sepsis, suggesting that microbiome disruption may be in the causal pathway and an independent risk factor. This study tests whether variation in the gut microbiota associates with risk of sepsis onset and its outcomes.MethodsUsing a validated surveillance definition, patients with an archived rectal swab from intensive care and hematology units were screened for sepsis. After confirmation by chart review, cases were matched to controls in a 1:2 ratio based on age, gender, and collection date. Relative taxon abundance was measured by sequence analysis of 16S rRNA gene amplicons; total bacterial abundance was measured by qPCR of the 23S rRNA gene. Conditional logistic regression identified clinical and microbiota variables associated with sepsis.ResultsThere were 103 sepsis cases matched to 206 controls. In a final model adjusting for exposure to broad-spectrum antibiotics and indwelling vascular catheters, high relative abundance (RA) of Enterococcus (Odds Ratio (OR) 1.36 per 10% increase, P=.016) and high total bacterial abundance (OR 1.50 per 10-fold increase in 23S copies/μL, P =.001) were independently associated with sepsis. Decreased RA of butyrate-producing bacteria also independently associated with sepsis (OR 1.20 for 10% decrease in RA, P =.041), and mortality in unadjusted analysis (OR=1.47 for 10% decrease in RA, P=.034).ConclusionsThis study indicates that the microbiota is altered at sepsis onset. The decreased RA of butyrate-producing bacteria in sepsis also associates with mortality, suggesting a therapeutic role for prebiotics and probiotics in the prevention and treatment of sepsis.ImportanceEarly detection of patients at risk for sepsis could enable interventions to prevent or rapidly treat this life-threatening condition. Prior antibiotic treatment is associated with sepsis, suggesting that disruption of the bacterial population in the gut (the intestinal microbiome) could be an important step leading to disease. To investigate this theory, we matched hospitalized patients with and without sepsis and characterized the patients’ microbiomes close to or at onset of sepsis. We found that several microbiome alterations, including having more total bacteria in the gut was associated with onset, regardless of prior antibiotic treatment. This signature of microbiome disruption brings us closer to identifying the biological causes of sepsis and could be used to develop new diagnostic tests to identify patients at risk of sepsis.

BackgroundThere is limited evidence on the performance of emergent large-vessel occlusion (LVO) stroke screening tools when used by emergency medical services (EMS) and emergency department (ED) providers. We assessed the validity and predictive value of the vision, aphasia, neglect (VAN) assessment when completed by EMS and in the ED among suspected stroke patients.MethodsWe conducted a retrospective study of VAN performed by EMS providers and VAN inferred from the National Institutes of Health Stroke Scale performed by ED nurses at a single hospital. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of VAN by EMS and in the ED for LVO and a combined LVO and intracerebral hemorrhage (ICH) outcome.ResultsFrom January 2018 to June 2020, 1,547 eligible patients were identified. Sensitivity and specificity of ED VAN were similar for LVO (72% and 74%, respectively), whereas EMS VAN was more sensitive (84%) than specific (68%). PPVs were low for both EMS VAN (26%) and ED VAN (21%) to detect LVO. Due to several VAN-positive ICHs, PPVs were substantially higher for both EMS VAN (44%) and ED VAN (39%) to detect LVO or ICH. EMS and ED VAN had high NPVs (97% and 96%, respectively).ConclusionsAmong suspected stroke patients, we found modest sensitivity and specificity of VAN to detect LVO for both EMS and ED providers. Moreover, the low PPV in our study suggests a significant number of patients with non-LVO ischemic stroke or ICH could be over-triaged with VAN.

This chapter discusses the histologic features of commonly encountered infections of the gastrointestinal (GI) tract and correlates the morphologic findings with microbiological and molecular testing. It also discusses the major differential diagnoses of infectious diseases of the GI tract that are encountered in practice.

We describe a case of chronic COVID-19 in a patient with lymphoma and associated B-cell immunodeficiency. Viral cultures and sequence analysis demonstrate ongoing replication of infectious SARS-CoV-2 virus for at least 119 days. The patient had three admissions related to COVID-19 over a four-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.

Increasing antibiotic resistance has resulted in infections that are life-threatening and difficult to treat. Interventions that prevent these infections, particularly without using antibiotics, could save lives. Intestinal colonization by pathogens, including vancomycin-resistant Enterococcus and carbapenem-resistant Enterobacteriaceae (part of the order Enterobacterales) is associated with subsequent infection, and increased colonization density is associated with increased infection risk. Therefore, colonization offers a window of opportunity for infection prevention if (i) there are rapid and inexpensive assays to detect colonization, (ii) there are safe and effective interventions, and (iii) the risk of infection outweighs the risk of the treatment. Fecal transplants are proof of principle that manipulating the microbiome can reduce such colonization and prevent infections. This study demonstrates the feasibility of implementing rapid and inexpensive assays to quantify colonization and measures the strength of association between Enterobacterales dominance and subsequent infection. The approach described here could be a valuable tool in the prevention of antibiotic-resistant infections., The Enterobacterales order of Gram-negative bacteria includes the common nosocomial pathogens Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Enterobacter species. Intestinal domination by some colonizing bacterial taxa is associated with subsequent infection, but 16S rRNA gene sequencing is too costly and slow to be used in a clinical setting. The objectives of this study were to develop a PCR-based assay that can measure Enterobacterales density, validate it against 16S rRNA gene sequencing, and measure the association between Enterobacterales dominance and subsequent infection. Two quantitative PCR (qPCR) assays that were developed to quantify the absolute and relative abundance of Enterobacterales had good correlation with 16S rRNA sequence analysis (P < 0.0001). Using both PCR assays and 16S sequencing, a matched case-control study was performed comparing rectal swabs from hospitalized patients who later developed bloodstream, urinary tract, or respiratory Enterobacterales infections (n = 95) to swabs from patients who remained uninfected (n = 189). Enterobacterales abundance measured by sequencing was high in both cases and controls (means, 31.1% and 27.5%, respectively; P = 0.322). We observed an increased risk of infection that depended on both the absolute and relative abundance of Enterobacterales as measured by qPCR assay A (P = 0.012). After adjustment for albumin levels, central venous catheter presence, and use of cephalosporins at the time of swab collection, this association still approached significance (P = 0.061). These results demonstrate that using qPCR to measure intestinal colonization dominance is feasible, indicate that hospitalized patients have high levels of Enterobacterales colonization, and suggest that both relative and absolute abundance may be associated with subsequent infection. IMPORTANCE Increasing antibiotic resistance has resulted in infections that are life-threatening and difficult to treat. Interventions that prevent these infections, particularly without using antibiotics, could save lives. Intestinal colonization by pathogens, including vancomycin-resistant Enterococcus and carbapenem-resistant Enterobacteriaceae (part of the order Enterobacterales) is associated with subsequent infection, and increased colonization density is associated with increased infection risk. Therefore, colonization offers a window of opportunity for infection prevention if (i) there are rapid and inexpensive assays to detect colonization, (ii) there are safe and effective interventions, and (iii) the risk of infection outweighs the risk of the treatment. Fecal transplants are proof of principle that manipulating the microbiome can reduce such colonization and prevent infections. This study demonstrates the feasibility of implementing rapid and inexpensive assays to quantify colonization and measures the strength of association between Enterobacterales dominance and subsequent infection. The approach described here could be a valuable tool in the prevention of antibiotic-resistant infections.

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 stymied 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 levels of CPS production and hmv, were all significantly out-competed by wildtype in a murine model of disseminating pneumonia. This suggests that an optimal balance between cellular energetics, CPS biosynthesis and hmv are required for maximal fitness. Altogether, these data demonstrate that hmv requires both CPS biosynthesis and other cellular factors, and that these processes are integrated into the metabolic status of the cell. Therefore, hvKp may require certain nutrients to fully elaborate its virulence-associated properties to specifically cause deep tissue infections.Author summaryKlebsiella pneumoniae is a common multi-drug resistant hospital-associated pathogen, however some isolates are capable of causing community-acquired infections in otherwise healthy individuals. The strains causing community-acquired infections have some distinguishing characteristics, which include overproduction of capsule and hypermucoviscosity. Hypermucoviscous strains are very tacky and sediment poorly when centrifuged. Historically, hypermucoviscosity has been attributed to overproduction of capsular polysaccharide, but recent data suggest that other factors contribute to this bacterial phenotype. Moreover, it seems that capsule and hypermucoviscosity may have distinct roles in pathogenesis. In this study, we sought to systematically investigate the genes that contribute to capsule and hypermucoviscosity. We found that in most cases, genes coordinately impact both capsule biosynthesis and hypermucoviscosity. Some metabolic genes linked to the TCA cycle, however, only affect one of these properties. Here, we identify that capsule biosynthesis and hypermucoviscosity are tightly tied to central metabolism and that an optimal balance between metabolism, capsule, and hypermucoviscosity are important for in vivo fitness of K. pneumoniae. These results identify genes that can be further probed to dissect how capsule and hypermucoviscosity are coordinated in response to niche-specific nutrients. Such studies will expand our understanding of the factors that drive the pathobiology of hypervirulent K. pneumoniae.

OBJECTIVES: The opioid crisis is a growing cause of mortality in the United States and may be mitigated by innovative approaches to identifying individuals at-risk of fatal opioid overdose. We examined Emergency Medical Services (EMS) utilization among a cohort of individuals who died from opioid overdose in order to identify potential opportunities for intervention. METHODS: Individuals who died of unintentional opioid overdose in a large North Carolina county between 01/01/2014 and 12/31/2016 were studied in a retrospective cohort. Death records obtained from North Carolina Vital Records were linked to EMS patient care records obtained from the county EMS System in order to describe the EMS encounters of each decedent in the year preceding their death. Patient demographics and EMS encounters were assessed to identify encounter characteristics that may be targeted for intervention. Chi-square tests and odds ratios were used to evaluate and characterize the statistical significance of differences in EMS utilization. RESULTS: Of the 218 individuals who died from unintentional opioid overdose in the study interval, 30% (n=66) utilized EMS in the year before their death and 17% (n=38) had at least one EMS encounter with documented drug or alcohol use (i.e. “drug-related encounter”). The mean age at death was 38 (range 19–74) years, 30% were female, 89% were White, and 8% were Black/African American. Factors associated with higher incidence of EMS utilization included age (P

Introductory paragraphGut colonization by the pathogen Klebsiella pneumoniae (Kp) is consistently associated with subsequent Kp disease1-5, and patients are predominantly infected with their colonizing strain1,2. However, colonizing strains likely vary in their potential to cause infection. We previously identified the plasmid-encoded tellurium resistance (ter) operon as highly associated with infection when compared to asymptomatic colonization in hospitalized patients1. The ter operon bestows resistance to the toxic compound tellurite oxide (TeO3−2), but this is unlikely to be its physiological function, as tellurium and TeO3−2 are exceedingly rare. Here we show that terC is necessary and terZABCDEF is sufficient for phenotypic TeO3−2 resistance. Next, we demonstrate that ter is encoded on a diverse group of plasmids without known plasmid-encoded virulence genes, suggesting an independent role in infection. Finally, our studies indicate that ter is a gut fitness factor, and its fitness advantage is conferred only when specific gut microbiota constituents are present. Collectively, these data reveal the Kp ter operon that is highly associated with human infection likely acts early in pathogenesis as a horizontally-transferrable fitness factor promoting robust gut colonization in the presence of the indigenous microbiota.

Macrophages are main effectors of heme metabolism, increasing transiently in the liver during heightened disposal of damaged or senescent red blood cells (sRBC). Macrophages are also essential in defense against microbial threats, but pathologic states of heme excess may be immunosuppressive. Herein, we uncovered a mechanism whereby an acute rise in sRBC disposal by macrophages led to an immunosuppressive phenotype following intrapulmonary Klebsiella pneumoniae infection characterized by increased extrapulmonary bacterial proliferation and reduced survival from sepsis in mice. The impaired immunity to K. pneumoniae during heightened sRBC disposal was independent of iron acquisition by bacterial siderophores, as K. pneumoniae mutant lacking siderophore function recapitulated findings observed with wildtype strain. Rather, sRBC disposal induced a liver transcriptomic profile notable for suppression of Stat1 and interferon-related responses during K. pneumoniae sepsis. Excess heme handling by macrophages recapitulated STAT1 suppression during infection that required synergistic NRF1 and NRF2 activation but was independent of heme oxygenase-1 induction. Whereas iron was dispensable, the porphyrin moiety of heme was sufficient to mediate suppression of STAT1-dependent responses in human and mouse macrophages and promoted liver dissemination of K. pneumoniae in vivo. Thus, cellular heme metabolism dysfunction negatively regulates the STAT1 pathway with implications in severe infection.

BackgroundPrior colonization by Klebsiella pneumoniae and vancomycin-resistant Enterococci (VRE) is associated with subsequent infection, particularly in intensive care unit (ICU) populations. Screening for VRE colonization, but not K. pneumoniae, is routinely performed in some health care systems. Identification of patient factors associated with K. pneumoniae colonization could enable infection prevention.MethodsICU patients were screened for VRE and K. pneumoniae by rectal swab culture over 2 time periods: July–October 2014 (n = 1209) and January–May 2016 (n = 1243). Patient demographics, baseline laboratory data, comorbidities, and outcomes were analyzed. 16S rRNA gene-based analysis was performed on a subset of patients (n = 248) to identify microbiota characteristics associated with VRE and K. pneumoniae colonization.ResultsK. pneumoniae colonization (17.3% of patients in the 2014 cohort, 7.3% in 2016) was significantly associated with VRE colonization in multivariable analysis (P = .03 in 2016; P = .08 in 2014). VRE colonization was associated with poor underlying health, whereas K. pneumoniae colonization was associated with advanced age. The most prevalent operational taxonomic units were Escherichia coli/Shigella spp., Klebsiella, and Enterococcus, consistent with high rates of detectable K. pneumoniae and VRE by culture. Microbial community structure in noncolonized patients was significantly different from those with VRE, K. pneumoniae, or both, attributable to differences in the relative abundance of Klebsiella and Enterococcus.ConclusionsK. pneumoniae co-colonizes with VRE and is a predominant taxon in ICU patients, but colonization was not associated with significant comorbidities. Screening for K. pneumoniae and VRE simultaneously could be an efficient approach for novel infection prevention strategies.

An integrated response to active threat events is essential to saving lives. Coordination of law enforcement officer (LEO) and emergency medical services (EMS) roles requires joint training, as maximizing survival is a shared responsibility. We sought to evaluate the performance of an integrated LEO-EMS Rescue Task Force (RTF) response to a simulated active shooter incident utilizing objective performance measures.Following prior didactic training, we conducted a series of evaluation scenarios for EMS providers and patrol officers in our urban/suburban advanced life support EMS system (pop. 1,000,000). The scenario-tested command staff, LEOs tasked with neutralizing an active shooter threat, and two RTFs of LEOs and EMS providers each tasked with triage and treatment of 11 simulated casualties scattered over 2 office building floors totaling 13,000 square feet. Trained evaluators recorded performance on 30 objective data elements related to LEO-EMS operations/communication, time intervals, and trauma care. Data were analyzed using descriptive statistics and t-tests for between group comparisons.Over 18 days, 69 scenario events evaluated 388 EMS providers and 468 LEOs. Overall median (90th percentile) times in minutes from dispatch were: unified command established 4.1 (5.5), RTF assembled 9.4 (13.5), first victim contact 11.9 (16.5), first victim to internal casualty collection point (CCP) 16.6 (20.8), all victims ready for evacuation 21.6 (26.0). Life-saving interventions included tourniquet placed: 96% (95% CI 92-99) and LEO placed tourniquet: 88% (79-94). Clinical delays included inappropriate chest decompression: 4% (2-9) and unnecessary initial treatment: 17% (12-25). Correct operational actions included communication with LEO to ensure EMS was safe to treat: 70% (61-77) and appropriate CCP selection: 84% (74-91). Incorrect operational actions included failure to maintain protective LEO-EMS formation: 49% (45-62) and inappropriate single patient evacuation: 20% (14-28). Limitations included the lack of a pre-training control group for this novel program.We described the performance of an integrated LEO-EMS Rescue Task Force response to a simulated active shooter event in a large city. In general, clinical care was appropriate while operational targets can be improved. Objective measurement of response goals may be used for benchmarking and performance improvement for active threat events.

Objective: The United States is currently experiencing a public health crisis of opioid overdoses. To determine where resources may be most needed, many public health officials utilize naloxone administration by EMS as an easily-measured surrogate marker for opioid overdoses in a community. Our objective was to evaluate whether naloxone administration by EMS accurately represents EMS calls for opioid overdose. We hypothesize that naloxone administration underestimates opioid overdose. Methods: We conducted a chart review of suspected overdose patients and any patients administered naloxone in Wake County, North Carolina, from January 2013 to December 2015. Patient care report narratives and other relevant data were extracted from electronic patient care records and the resultant database was analyzed by two EMS physicians. Cases were divided into categories including “known opioid use,” “presumed opioid use,” “no known opioid,” “altered mental status,” “cardiac arrest with known opioid use,” “cardiac arrest with no known opioid use,” or “suspected alcohol intoxication,” and then further separated based on whether naloxone was administered. Patient categories were compared by patient demographics and incident year. Using the chart review classification as the gold standard, we calculated the sensitivity and positive predictive value (PPV) of naloxone administration for opioid overdose. Results: A total of 4,758 overdose cases from years 2013–15 were identified. During the same period, 1,351 patients were administered naloxone. Of the 1,431 patients with known or presumed opioid use, 57% (810 patients) received naloxone and 43% (621 patients) did not. The sensitivity of naloxone administration for the identification of patients with known or presumed opioid use was 57% (95% CI: 54%–59%) and the PPV was 60% (95% CI: 57%–63%). Conclusion: Among patients receiving care in this large urban EMS system in the United States, the overall sensitivity and positive predictive value for naloxone administration for identifying opioid overdoses was low. Better methods of identifying opioid overdose trends are needed to accurately characterize the burden of opioid overdose within and among communities.

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., Author summary The bacteria Klebsiella pneumoniae (Kp) is an important cause of infection in healthcare settings. These infections can be difficult to treat, as they frequently occur in chronically ill patients and the bacteria have the ability to acquire multiple antibiotic resistance markers. Kp is a common colonizer of the intestinal tract in hospitalized patients, and can progress to infections of the bloodstream, respiratory, and urinary tract. However, the bacterial factors that allow Kp to replicate in these different body sites are unclear. In this study, we found that the Kp citrate synthase, GltA, enables bacterial replication in the lung and intestine by enhancing the ability of Kp to use diverse nutrients in a mechanism known as metabolic flexibility. Kp lacking GltA require specific amino acids that are abundant in blood, but not other body sites. The work in this study provides novel insight into why Kp is a successful hospital pathogen that can colonize and infect multiple body sites.

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, including the innate immune protein lipocalin 2 (Lcn2) that prevents Kp iron acquisition by sequestering the siderophore enterobactin. To identify novel 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 toLcn2+/+andLcn2-/-mice. Comparing mutant frequencies between mouse genotypes, this genome-wide screen identified the Kp citrate synthase GltA as potentially interacting with Lcn2, and this novel finding was independently validated. Interestingly,in vitrostudies 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 thegltAmutant has a distinct amino acid auxotrophy and is unable to use a variety of carbon sources. Specifically, we show thatgltAis necessary for growth in bronchioloalveolar lavage fluid, which is amino acid-limited, but dispensable in serum, which is amino acid rich. Deletion ofLcn2from the host leads to increased amino acid levels in bronchioloalveolar lavage fluid, and thus abrogates the loss ofgltAduring pneumonia in theLcn2-/-background. GltA was also required for gut colonization, but dispensable in the bloodstream in a bacteremia model, demonstrating that deletion ofgltAleads to an organ-specific fitness defect. Together, this study is the first to mechanistically describe a role forgltAin Kp infection and provide unique insight into how metabolic flexibility impacts bacterial fitness during infection.Author SummaryThe bacteriaKlebsiella pneumoniae(Kp) is an important cause of infection in healthcare settings. These infections can be difficult to treat, as they frequently occur in chronically ill patients and the bacteria has the ability to acquire multiple antibiotic resistance markers. Kp is a common colonizer of the intestinal tract in hospitalized patients, and can progress to infections of the bloodstream, respiratory and urinary tract. However, the bacterial factors that allow Kp to replicate in these different body sites is unclear. In this study, we found that the Kp citrate synthase, GltA, enables bacterial replication in the lung and intestine by enhancing the ability of Kp to use diverse nutrients, in a mechanism known as metabolic flexibility. Kp lacking GltA require specific amino acids that are abundant in blood, but not other body sites. The work in this study provides novel insight into why Kp is a successful hospital pathogen that can colonize and infect multiple body sites.

Key Clinical Message Disseminated histoplasmosis and hemophagocytic lymphohistiocytosis show overlapping features, which require careful contextual interpretation. Histopathologic evaluation can potentially rapidly identify cases of possible histoplasmosis. A high index of clinical suspicion, particularly in endemic areas and in a setting of immunosuppression, is critical to appropriate diagnosis and treatment.