Background Klebsiella pneumoniae strains have been divided into two major categories: classical K. pneumoniae, which are frequently multidrug-resistant and cause hospital-acquired infections in patients with impaired defenses, and hypervirulent K. pneumoniae, which cause severe community-acquired and disseminated infections in normal hosts. Both types of infections may lead to bacteremia and are associated with significant morbidity and mortality. The relative burden of these two types of K. pneumoniae among bloodstream isolates within the United States is not well understood. Methods We evaluated consecutive K. pneumoniae isolates cultured from the blood of hospitalized patients at Northwestern Memorial Hospital (NMH) in Chicago, Illinois between April 2015 and April 2017. Bloodstream isolates underwent whole genome sequencing, and sequence types (STs), capsule loci (KLs), virulence genes, and antimicrobial resistance genes were identified in the genomes using the bioinformatic tools Kleborate and Kaptive. Patient demographic, comorbidity, and infection information, as well as the phenotypic antimicrobial resistance of the isolates were extracted from the electronic health record. Candidate hypervirulent isolates were tested in a murine model of pneumonia, and their plasmids were characterized using long-read sequencing. We also extracted STs, KLs, and virulence and antimicrobial resistance genes from the genomes of bloodstream isolates submitted from 33 United States institutions between 2007 and 2021 to the National Center for Biotechnology Information (NCBI) database. Results Consecutive K. pneumoniae bloodstream isolates (n = 104, one per patient) from NMH consisted of 75 distinct STs and 51 unique capsule loci. The majority of these isolates (n = 58, 55.8%) were susceptible to all tested antibiotics except ampicillin, but 17 (16.3%) were multidrug-resistant. A total of 32 (30.8%) of these isolates were STs of known high-risk clones, including ST258 and ST45. In particular, 18 (17.3%) were resistant to ceftriaxone (of which 17 harbored extended-spectrum beta-lactamase genes) and 9 (8.7%) were resistant to meropenem (all of which harbored a carbapenemase genes). Four (3.8%) of the 104 isolates were hypervirulent K. pneumoniae, as evidenced by hypermucoviscous phenotypes, high levels of virulence in a murine model of pneumonia, and the presence of large plasmids similar to characterized hypervirulence plasmids. These isolates were cultured from patients who had not recently traveled to Asia. Two of these hypervirulent isolates belonged to the well characterized ST23 lineage and one to the re-emerging ST66 lineage. Of particular concern, two of these isolates contained plasmids with tra conjugation loci suggesting the potential for transmission. We also analyzed 963 publicly available genomes of K. pneumoniae bloodstream isolates from locations within the United States. Of these, 465 (48.3%) and 760 (78.9%) contained extended-spectrum beta-lactamase genes or carbapenemase genes, respectively, suggesting a bias towards submission of antibiotic-resistant isolates. The known multidrug-resistant high-risk clones ST258 and ST307 were the predominant sequence types. A total of 32 (3.3%) of these isolates contained aerobactin biosynthesis genes and 26 (2.7%) contained at least two genetic features of hvKP strains, suggesting elevated levels of virulence. We identified 6 (0.6%) isolates that were STs associated with hvKP: ST23 (n = 4), ST380 (n = 1), and ST65 (n = 1). Conclusions Examination of consecutive isolates from a single center demonstrated that multidrug-resistant high-risk clones are indeed common, but a small number of hypervirulent K. pneumoniae isolates were also observed in patients with no recent travel history to Asia, suggesting that these isolates are undergoing community spread in the United States. A larger collection of publicly available bloodstream isolate genomes also suggested that hypervirulent K. pneumoniae strains are present but rare in the USA; however, this collection appears to be heavily biased towards highly antibiotic-resistant isolates (and correspondingly away from hypervirulent isolates).

are current full-time employees of MicroB-plex, Inc.Geena Kim and Shant Ohanian are former full-time employees of MicroB-plex, Inc., and generated samples and data reported in this manuscript. F. Eun-Hyung Lee, M.D., is the founder of MicroB-plex, Inc, and an Associate Professor at Emory University. She did not draw salary on this project, but she was actively engaged in grant-writing, project administration and production of the manuscript. John L. Daiss, Ph.D., works more than half-time for MicroB-plex, Inc. and also serves as a Research Associate Professor at the University of Rochester Medical Center. L. Edward Cannon was the CEO of MicroBplex, Inc., throughout the time of this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Pseudomonas aeruginosa is a frequent cause of antibiotic-resistant infections. Although P. aeruginosa is intrinsically resistant to many antimicrobial agents, aminoglycosides are active against this organism in the absence of acquired resistance determinants and mutations. However, genes encoding aminoglycoside modifying enzymes (AMEs) are found in many strains that are resistant to these agents. We examined the prevalence of phenotypic resistance to the commonly used aminoglycosides gentamicin, tobramycin, and amikacin in a collection of 227 P. aeruginosa bloodstream isolates collected over two decades from a single U.S. academic medical institution. Resistance to these antibiotics was relatively stable over this time period. High-risk clones ST111 and ST298 were initially common but decreased in frequency over time. Whole genome sequencing identified relatively few AME genes in this collection compared to the published literature; only 14% of isolates contained an AME gene other than the ubiquitous aph(3’)-IIb. Of those present, only ant(2”)-Ia was associated with phenotypic resistance to gentamicin or to tobramycin. One extensively drug-resistant strain, PS1871, contained 5 AME genes, most of which were part of clusters of antibiotic resistance genes embedded within transposable elements. These findings suggest that AME genes play a relatively minor role in aminoglycoside resistance at our institution but that multidrug-resistant strains remain a problem.

Background: Within eight weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection. This study demonstrates the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Methods: Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Serum and a novel matrix from circulating plasmablasts called " M edium E nriched for N ewly S ynthesized A ntibodies" (MENSA) samples were collected during weeks 1, 2, and 4. Antibodies specific for ten C. difficile antigens were measured in each sample Results: Among the 46 C. difficile -infected patients, nine (19.5%) experienced recurrence within eight weeks of primary infection. Among the 37 non-recurrent patients, 23 (62%; 23/37) had anti- C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred early (within the first 12 days post-symptom onset), including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (51%; 19/37). In contrast, none (0%; 0/9) of the patients who subsequently recurred after hospitalization produced antibodies specific for any of the three C. difficile toxin antigens. Thus, patients with a negative early MENSA response against all three C. difficile toxin antigens had a 19-fold greater relative risk of recurrence. Discussion: MENSA and serum levels of IgA and/or IgG antibodies for three C. difficile toxins have prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence thereby providing a biomarker of protection from recurrent CDI. Patients who are positive by this immunoassay are unlikely to suffer recurrence. Early identification of patients at-risk for recurrence by negative MENSA creates opportunities for targeted prophylactic strategies that can reduce the incidence, cost and morbidity due to recurrent CDI.

47BACKGROUND. Clostridioides difficile infections (CDI) have been a challenging and 48 increasing serious concern in recent years. While early and accurate diagnosis is crucial, available assays 49 have frustrating limitations 50 51 OBJECTIVE. Develop a simple, blood-based immunoassay to accurately diagnose patients 52 suffering from active CDI. 53 54 MATERIALS AND METHODS. Uninfected controls (n=95) and CDI patients (n=167) were 55 recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a 56 positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly 57 synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. 58 difficile antigens. 59 60 RESULTS. Sixty-six percent of CDI patients produced measurable responses to C. difficile 61 antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI 62 patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in 63 MENSA, and 27/167 (16%) were detectable only in serum. 64 65 DISCUSSION. We describe the results of a multiplex immunoassay for the diagnosis of ongoing 66 CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, 67 serum-positive only, MENSA-and serum-positive, and MENSA-and serum-negative. The MENSA 68 positive-only patients accounted for 30% and may be attributed to nascent antibody secretion in MENSA 69 prior to seroconversion. Conversely, the serum positive-only subset may have been more advanced in 70 5 5 their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI 71 patients who were not identified using MENSA or serum immunoassays. 72 73 IMPORTANCE. While there was considerable overlap between patients identified through 74 MENSA and serum, both methods detected additional, unique patients. The combined use of both 75 MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, 76 longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host 77 humoral immune responses in CDI patients. 78In this observational investigation, we present the results of a pilot study validating the use of a 127 multi-antigen immunoassay and its ability to measure antibody secretion reflecting ongoing CDI in 128 MENSA samples from a complex hospital population and compare its performance with results from sera. 129 130 RESULTS 131

BackgroundWithin eight weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no validated biomarkers predictive of recurrence during primary infection. This study demonstrates the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence.MethodsForty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Serum and MENSA samples prepared during weeks 1, 2, and 4 following symptom-onset were measured for antibodies specific for ten C. difficile antigens.ResultsAmong the 46 C. difficile-infected patients, nine (19.5%) experienced recurrence within eight weeks of primary infection. Among the 37 non-recurrent patients, 23 had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred within the first week post-symptom onset, including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (19/37). In contrast, none of the patients who subsequently recurred after hospitalization produced antibodies specific for the three C. difficile toxin antigens. IgA antibodies for the toxin antigens demonstrated the greatest predictive power for protection from recurrence.DiscussionThe development of IgG and/or IgA antibodies for three C. difficile toxins in serum and/or MENSA has prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence. Early identification of patients at-risk for recurrence can reduce costs and morbidity.

Pseudomonas aeruginosa is frequently resistant to multiple antibiotics, including aminoglycosides. The rates of resistance to aminoglycosides in bloodstream isolates collected over 2 decades at a United States hospital remained constant, suggesting that antibiotic stewardship programs may be effective in countering an increase in resistance.

Background P. aeruginosa is a cause of hospital-acquired and ventilator-associated pneumonia. Hypermutator P. aeruginosa strains have been described in patients with cystic fibrosis and chronic respiratory infections but are rare in patients with acute P. aeruginosa infection. This case describes a hypermutator strain of P. aeruginosa found in a patient with COVID-19-associated acute respiratory distress syndrome (ARDS). Methods Serial respiratory and blood cultures were collected. Short-read sequencing libraries were prepared using the Illumina Nextera XT kit, and whole-genome sequencing was performed using the Illumina NextSeq platform. Long-read sequencing libraries were prepared from unsheared genomic DNA using ligation sequencing kit SQK-LSK109 and sequenced on the Oxford MinION platform. Single nucleotide variants were identified by aligning reads from each isolate to the complete genome of the first available clinical isolate. Hypermutator assays were performed by measuring the mutation frequency rate for rifampin resistance. Antibiotic minimal inhibitory concentrations (MICs) were performed. Growth curves were performed with a starting OD600 of 0.1 with measurements taken every 30 minutes for 24 hours. Results Seventeen respiratory and five blood isolates were obtained throughout 62 days of hospitalization. Fourteen of the 22 isolates exhibited hypermutator phenotypes by rifampin resistance assays, which demonstrated rapid accumulation of mutations. All five bloodstream isolates were hypermutators. MIC testing noted increased resistance to aminoglycosides, fluoroquinolones, and aztreonam in the hypermutator isolates. All bloodstream isolates descended from a single progenitor noted on whole-genome sequencing. Each hypermutator strain contained a mutation in the mismatch repair gene mutL, previously associated with the hypermutator phenotype. Genetic Tree of Patient Isolates The genetic tree highlights hypermutator versus non-hypermutator single nucleotide variants Conclusion This case was notable for multiple isolates of hypermutator P. aeruginosa that persisted over weeks. The patient’s COVID-19 infection and acute respiratory distress syndrome may have facilitated persistence of the P. aeruginosa lineage, allowing a hypermutator lineage to emerge. Disclosures All Authors: No reported disclosures.