Background Klebsiella pneumoniae is a leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive K pneumoniae. We sought to determine whether the O-specific polysaccharide antigen is immunogenic in humans with K pneumoniae bloodstream infection. We also sought to define the cross-reactivity of human antibody responses among structurally related K pneumoniae O-specific polysaccharide subtypes and to assess the effect of capsule production on O-specific polysaccharide-targeted antibody binding and function. Methods In this prospective cohort study, we compared plasma antibody responses to O-specific polysaccharide in a cohort of consecutively enrolled patients with K pneumoniae bloodstream infection with controls, specifically a cohort of healthy individuals and a cohort of individuals with Enterococcus spp bloodstream infection. Patients were enrolled at the Massachusetts General Hospital, a tertiary hospital with affiliated clinics in the USA. We excluded patients whose isolates were not confirmed to be K pneumoniae by whole-genome sequencing. The primary outcome was the measurement of plasma IgG, IgM, and IgA antibody responses. We performed flow cytometry to measure the effects of K pneumoniae capsule production on O-specific polysaccharide antibody binding and O-specific polysaccharide antibody-mediated complement deposition, using patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. Findings We enrolled 129 consecutive patients with suspected K pneumoniae bloodstream infection between July 24, 2021, and August 4, 2022, of whom 69 patients (44 [64%] male and 25 [36%] female) with confirmed K pneumoniae bloodstream infection were eligible for immunological evaluation. Common O-specific polysaccharide serotypes (O1, O2, O3, and O5) accounted for 57 (83%) of 69 infections. O-specific polysaccharide was immunogenic in patients with K pneumoniae bloodstream infection, and peak O-specific polysaccharide-IgG antibody responses in patients were ten-fold to 30-fold higher than antibody responses detected in healthy controls, depending on the serotype. There was cross-reactivity among similar O-specific polysaccharide subtypes, including the O1v1 and O1v2, O2v1 and O2v2, and O3 and O3b subtypes, as well as between the O1 and O2 types. Capsule produced by both hyperencapsulated and non-hyperencapsulated K pneumoniae inhibited O-specific polysaccharide-targeted antibody binding and function. Interpretation O-specific polysaccharide was immunogenic in patients with K pneumoniae bloodstream infection, supporting its potential as a candidate vaccine antigen. The cross-reactivity observed between similar O-specific polysaccharide subtypes in patients with K pneumoniae bloodstream infection suggests that it might not be necessary to include all subtypes in an O-specific polysaccharide-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with O-specific polysaccharide antibody binding. This finding could limit the effectiveness of vaccines that exclusively target O-specific polysaccharide. Funding National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

Background. The clinical and microbial factors associated with Klebsiella pneumoniae bloodstream infections (BSIs) are not well characterized. Prior studies have focused on highly resistant or hypervirulent isolates, limiting our understanding of K. pneumoniae strains that commonly cause BSI. We performed a record review and whole-genome sequencing to investigate the clinical characteristics, bacterial diversity, determinants of antimicrobial resistance, and risk factors for in-hospital death in a cohort of patients with K. pneumoniae BSI.Methods. We identified 562 patients at Massachusetts General Hospital with K. pneumoniae BSIs between 2016 and 2022. We collected data on comorbid conditions, infection source, clinical outcomes, and antibiotic resistance and performed whole-genome sequencing on 108 sequential BSI isolates from 2021 to 2022.Results. Intra-abdominal infection was the most common source of infection accounting for 34% of all BSIs. A respiratory tract source accounted for 6% of BSIs but was associated with a higher in-hospital mortality rate (adjusted odds ratio, 5.4 [95% confidence interval, 2.2-12.8]; P < .001 for comparison with other sources). Resistance to the first antibiotic prescribed was also associated with a higher risk of death (adjusted odds ratio, 5.2 [95% confidence interval, 2.2-12.4]; P < .001). BSI isolates were genetically diverse, and no clusters of epidemiologically and genetically linked cases were observed. Virulence factors associated with invasiveness were observed at a low prevalence, although an unexpected association between O-antigen type and the source of infection was found.Conclusions. These observations demonstrate the versatility of K. pneumoniae as an opportunistic pathogen and highlight the need for new approaches for surveillance and the rapid identification of patients with invasive antimicrobial-resistant K. pneumoniae infection.

Abstract Background Antimicrobial resistant (AMR) bacterial infections pose a significant and growing health threat worldwide. The burden of these resistant infections is often most severe in lower income areas, where the medical infrastructure necessary for their diagnosis is lacking. The development of cost-effective and accessible diagnostic tools for the rapid identification of resistant infections in low-resource settings is crucial. Specific High-sensitivity Enzymatic Reporter Unlocking, or SHERLOCK, is a promising CRISPR-based system that can aid in the diagnosis of infections and support clinical decision making. Here, we present an assay termed BADLOCK (Bacteria and AMR Detection by SHERLOCK) that uses SHERLOCK technology to rapidly identify bacterial species and AMR genes on blood cultures. SHERLOCK Schematic Enzymatic activity of the Cas protein is turned on when the target gene is bound, which then activates reporter probes. Methods We combine isothermal amplification of gene targets with SHERLOCK-based detection into a single step, allowing for a streamlined workflow and a total time-to-detection of less than two hours. The assay targets the topA gene for species identification, which is highly conserved within species but has sufficient variability between them to allow for discrimination between even closely related pathogens. A panel of clinically important AMR genes, including CTX-M-15, mecA, and five canonical carbapenemases, is also targeted to provide information on resistance profiles. BADLOCK Targets Gene targets included in the BADLOCK assay. Results BADLOCK has high sensitivity and specificity for both AMR genes and species identification and leverages a lateral-flow platform to provide a simple visual readout. We are currently validating BADLOCK on a library of several hundred clinical isolates with known genetic backgrounds, including a large collection of carbapenem-resistant Enterobacterales, which are major contributors to the global burden of resistant infections. Lateral Flow Assay Results Lateral flow results. Strip 1 is the topA gene specific to K. pneumoniae. Strips 2-4 are AMR genes KPC, CTX-M-15, and MecA. Lane 5 is a positive control; lane 6 is a NTC with bottom banding. “+” and “-” symbols mark representative readouts. Conclusion We have developed an assay for the cheap, rapid detection of bacterial infections of the bloodstream capable of detecting common pathogens as well as a panel of AMR genes. Our work has the potential to improve the diagnostic landscape for resistant infections in low-and-middle-income countries, which would represent a major step forward in infectious disease treatment in these underserved areas. Disclosures All Authors: No reported disclosures

BACKGROUND:COVID-19 has a widely variable clinical syndrome that is difficult to distinguish from bacterial sepsis, leading to high rates of antibiotic use. Early studies indicate low rates of secondary bacterial infections (SBIs) but have included heterogeneous patient populations. Here, we catalogue all SBIs and antibiotic prescription practices in a population of mechanically ventilated patients with COVID-19 induced acute respiratory distress syndrome (ARDS).METHODS:This was a retrospective cohort study of all patients with COVID-19 ARDS requiring mechanical ventilation from 3 Seattle, Washington hospitals in 2020. Data were obtained via electronic and manual review of the electronic medical record. We report the incidence and site of SBIs, mortality, and antibiotics per day using descriptive statistics.RESULTS:We identified 126 patients with COVID-19 induced ARDS during the study period. Of these patients, 61% developed clinical infection confirmed by bacterial culture. Ventilator associated pneumonia was confirmed in 55% of patients, bacteremia in 20%, and urinary tract infection (UTI) in 17%. Staphylococcus aureus was the most commonly isolated bacterial species. A total of 97% of patients received antibiotics during their hospitalization, and patients received nearly one antibiotic per day during their hospital stay.CONCLUSIONS:Mechanically ventilated patients with COVID-19 induced ARDS are at high risk for secondary bacterial infections and have extensive antibiotic exposure.

Background. Klebsiella pneumoniae is a bacterial pathogen with increasing rates of resistance to carbapenem antibiotics, but the population structure and genetic drivers of carbapenem-resistant K pneumoniae (CRKP) remain underexplored in developing countries. Carbapenem-resistant K pneumoniae were recently introduced into Peru but have grown rapidly in prevalence, enabling study of this pathogen as it expands into an unaffected environment. Methods. In this study, using whole genome sequencing, we show that 3 distinct lineages encompass almost all CRKP identified in the hospital where it was first reported in Peru. Results. The most prevalent lineage, ST348, has not been described outside of Europe, raising concern for global dissemination. We identified metallo-beta -lactamase NDM-1 as the primary carbapenem resistance effector, which was harbored on a novel vector resulting from recombination between 2 different plasmids, pKPI -NDM-1 and pMS7884A. Conclusions. This study is the first of its kind performed in Peru, and it furthers our understanding of the landscape of CRKP infections in Latin America.

Large-scale bacterial genome sequencing efforts to date have provided limited information on the most prevalent category of disease: sporadically acquired infections caused by common pathogenic bacteria. Here, we performed whole-genome sequencing and de novo assembly of 312 blood- or urine-derived isolates of extraintestinal pathogenic (ExPEC) Escherichia coli, a common agent of sepsis and community-acquired urinary tract infections, obtained during the course of routine clinical care at a single institution. We find that ExPEC E. coli are highly genomically heterogeneous, consistent with pan-genome analyses encompassing the larger species. Investigation of differential virulence factor content and antibiotic resistance phenotypes reveals markedly different profiles among lineages and among strains infecting different body sites. We use high-resolution molecular epidemiology to explore the dynamics of infections at the level of individual patients, including identification of possible person-to-person transmission. Notably, a limited number of discrete lineages caused the majority of bloodstream infections, including one subclone (ST131-H30) responsible for 28% of bacteremic E. coli infections over a 3-yr period. We additionally use a microbial genome-wide-association study (GWAS) approach to identify individual genes responsible for antibiotic resistance, successfully recovering known genes but notably not identifying any novel factors. We anticipate that in the near future, whole-genome sequencing of microorganisms associated with clinical disease will become routine. Our study reveals what kind of information can be obtained from sequencing clinical isolates on a large scale, even well-characterized organisms such as E. coli, and provides insight into how this information might be utilized in a healthcare setting.

Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital's intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care.

The PRDM9 gene encodes a protein with a highly variable tandem-repeat zinc finger (ZF) DNA-binding domain that plays a key role in determining sequence-specific hotspots of meiotic recombination genome wide. Here we survey the diversity of the PRDM9 ZF domain by sequencing this region in 64 primates from 18 species, revealing 68 unique alleles across all groups. We report ubiquitous positive selection at nucleotide positions corresponding to DNA contact residues and the expansion of ZFs within clades, which confirms the rapid evolution of the ZF domain throughout the primate lineage. Alignment of Neandertal and Denisovan sequences suggests that PRDM9 in archaic hominins was closely related to present-day human alleles that are rare and specific to African populations. In the context of its role in reproduction, our results are consistent with variation in PRDM9 contributing to speciation events in primates.