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ɑ/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) ɑ and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (T hroughput- I ntensive R apid T CR L ibrary seq uencing ) , a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both ɑ and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.

SARS-CoV-2 has caused millions of infections worldwide since its emergence in 2019. Understanding how infection and vaccination induce mucosal immune responses and how they fluctuate over time is important, especially since they are key in preventing infection and reducing disease severity. We established a novel methodology for assessing SARS-CoV-2 cytokine and antibody responses at the nasal epithelium by using nasopharyngeal swabs collected longitudinally before and after either SARS-CoV-2 infection or vaccination. We then compared responses between mucosal and systemic compartments. We demonstrate that cytokine and antibody profiles differ between compartments. Nasal cytokines show a wound healing phenotype while plasma cytokines are consistent with pro-inflammatory pathways. We found that nasal IgA and IgG have different kinetics after infection, with IgA peaking first. Although vaccination results in low nasal IgA, IgG induction persists for up to 180 days post-vaccination. This research highlights the importance of studying mucosal responses in addition to systemic responses to respiratory infections. The methods described herein can be used to further mucosal vaccine development by giving us a better understanding of immunity at the nasal epithelium providing a simpler, alternative clinical practice to studying mucosal responses to infection., (© 2023. Springer Nature Limited.)

Evidence suggests that innate and adaptive cellular responses mediate resistance to the influenza virus and confer protection after vaccination. However, few studies have resolved the contribution of cellular responses within the context of preexisting antibody titers. Here, we measured the peripheral immune profiles of 206 vaccinated or unvaccinated adults to determine how baseline variations in the cellular and humoral immune compartments contribute independently or synergistically to the risk of developing symptomatic influenza. Protection correlated with diverse and polyfunctional CD4 + and CD8 + T, circulating T follicular helper, T helper type 17, myeloid dendritic and CD16 + natural killer (NK) cell subsets. Conversely, increased susceptibility was predominantly attributed to nonspecific inflammatory populations, including γδ T cells and activated CD16 - NK cells, as well as TNFα + single-cytokine-producing CD8 + T cells. Multivariate and predictive modeling indicated that cellular subsets (1) work synergistically with humoral immunity to confer protection, (2) improve model performance over demographic and serologic factors alone and (3) comprise the most important predictive covariates. Together, these results demonstrate that preinfection peripheral cell composition improves the prediction of symptomatic influenza susceptibility over vaccination, demographics or serology alone., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Piperacillin-tazobactam (TZP) is frequently used for intra-abdominal infection (IAI). Our institution experienced consecutive shortages of TZP and cefepime, providing an opportunity to review prescribing patterns and microbiology for IAI. Hospitalized adult patients treated for IAI, based on provider selection of IAI as the indication within the antibiotic order, between March 2014 and February 2018 were identified from the University of Virginia Clinical Data Repository and Infection Prevention and Control Database. Antimicrobial utilization, microbiologic data, and clinical outcomes were compared across four year-long periods: pre-shortage, TZP shortage, cefepime shortage, and post-shortage. There were 7,668 episodes of antimicrobial prescribing for an indication of IAI during the study period. Cefepime use for IAI increased 190% during the TZP shortage; meanwhile ceftriaxone use increased by only 57%. There was no increase in in-house mortality, colonization with resistant organisms, or Clostridiodes difficile infection among patients treated with IAI during the shortage periods. Among a subset of cases randomly selected for review, Pseudomonas sp. was a rare cause of IAI, but anti-pseudomonal antibiotics were commonly prescribed empirically. We observed a large increase in cefepime utilization for IAI during a TZP shortage that was not warranted based on the observed frequency of identification of Pseudomonas sp. as the causative organism in IAI, suggesting a need to revisit national guideline recommendations., (Copyright © 2021 Park et al.)

Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7 - CD45RA + effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hospital wastewater is an increasingly recognized reservoir for resistant Gram-negative organisms. Factors involved in establishment and persistence of Klebsiella pneumoniae carbapenemase-producing organisms (KPCOs) in hospital wastewater plumbing are unclear. This study was conducted at a hospital with endemic KPCOs linked to wastewater reservoirs and robust patient perirectal screening for silent KPCO carriage. Over 5 months, both rooms occupied and rooms not occupied by KPCO-positive patients were sampled at three wastewater sites within each room (sink drain, sink P-trap, and toilet or hopper). Risk factors for KPCO positivity were assessed using logistic regression. Whole-genome sequencing (WGS) identified environmental seeding by KPCO-positive patients. A total of 219/475 (46%) room sampling events were KPCO positive in at least one wastewater site. KPCO-positive patient exposure was associated with increased risk of environmental positivity for the room and toilet/hopper. Previous positivity and intensive care unit room type were consistently associated with increased risk. Tube feeds were associated with increased risk for the drain, while exposure to patients with Clostridioides difficile was associated with decreased risk. Urinary catheter exposure was associated with increased risk of P-trap positivity. P-trap heaters reduced risk of P-trap and sink drain positivity. WGS identified genomically linked environmental seeding in 6 of 99 room occupations by 40 KPCO-positive patients. In conclusion, KPCO-positive patients seed the environment in at least 6% of opportunities; once positive for KPCOs, wastewater sites are at greater risk of being positive subsequently. Increased nutrient exposure, e.g., due to tube food disposal down sinks, may increase risk; frequent flushing may be protective. IMPORTANCE Klebsiella pneumoniae carbapenemase-producing organisms (KPCOs) are bacteria that are resistant to most antibiotics and thus are challenging to treat when they cause infections in patients. These organisms can be acquired by patients who are hospitalized for other reasons, complicating their hospital stay and even leading to death. Hospital wastewater sites, such as sink drains and toilets, have played a role in many reported outbreaks over the past decade. The significance of our research is in identifying risk factors for environmental positivity for KPCOs, which will facilitate further work to prevent transmission of these organisms to patients from the hospital environment., (Copyright © 2020 Park et al.)

With multidrug-resistant (MDR) Enterobacterales on the rise, a nontoxic antimicrobial agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non- Escherichia coli isolates in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with 96 KPC-producing clinical isolates of multiple strains and species collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole-genome sequencing (WGS) data using NCBI's AMRFinder and custom HMM search. Susceptibility testing was performed using a glucose-6-phosphate-supplemented fosfomycin Etest and Kirby-Bauer disk diffusion (DD) assays, and the results were compared to those obtained by agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by Etest, 70% (67/96) by DD, and 88% (84/96) by agar dilution. fosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA -positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA positive compared to those that were fosA negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes., (Copyright © 2019 Elliott et al.)

The recent development of new antimicrobials active against carbapenemase-producing Enterobacteriales (CPE) has brought new hope for the treatment of infections due to these organisms. However, the evolving epidemiology of bacteria with carbapenemases may complicate management, as providers are faced with treating patients colonized by bacteria producing multiple carbapenemases. Here, we present the clinical course and treatment of Raoultella planticola bacteremia in a cirrhotic patient known to be colonized with both bla KPC - and bla OXA-48 -carrying organisms., (Copyright © 2019 American Society for Microbiology.)

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, bla KPC , between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 bla KPC -positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate bla KPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae ( n  = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique bla KPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases., (Copyright © 2019 Mathers et al.)

As the spread of antimicrobial resistance (AMR) genes becomes an increasing global threat, improved understanding of mobile genetic elements which contribute to the spread of antimicrobial resistance genes, becomes more critical. We created transconjugants from the mating of three chromosomally isogenic Klebsiella pneumoniae carbapenemase (bla KPC ) positive Citrobacter freundii isolates with a laboratory strain of Escherichia coli and evaluated the movement of small cryptic plasmids (SCPs), p3223 and p1916, when larger bla KPC -plasmids were transferred. In all of the 143 transconjugants, multiple plasmids, both large and small, transferred with each mating. When two bla KPC- plasmids were present in the host, frequently (87%; 98/113) both would be transferred during mating. p3223 is found in a wide range of bacterial hosts that harbor AMR genes; p1916 has been identified in only a limited number of publicly available sequences to date. From our evaluation, there is still much to learn about SCPs, and the high rate of co-transfer of multiple plasmids from real-world carbapenemase-producing Enterobacteriales., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Much of the worldwide dissemination of antibiotic resistance has been driven by resistance gene associations with mobile genetic elements (MGEs), such as plasmids and transposons. Although increasing, our understanding of resistance spread remains relatively limited, as methods for tracking mobile resistance genes through multiple species, strains and plasmids are lacking. We have developed a bioinformatic pipeline for tracking variation within, and mobility of, specific transposable elements (TEs), such as transposons carrying antibiotic-resistance genes. TETyper takes short-read whole-genome sequencing data as input and identifies single-nucleotide mutations and deletions within the TE of interest, to enable tracking of specific sequence variants, as well as the surrounding genetic context(s), to enable identification of transposition events. A major advantage of TETyper over previous methods is that it does not require a genome reference. To investigate global dissemination of Klebsiella pneumoniae carbapenemase (KPC) and its associated transposon Tn4401, we applied TETyper to a collection of over 3000 publicly available Illumina datasets containing blaKPC. This revealed surprising diversity, with over 200 distinct flanking genetic contexts for Tn4401, indicating high levels of transposition. Integration of sample metadata revealed insights into associations between geographic locations, host species, Tn4401 sequence variants and flanking genetic contexts. To demonstrate the ability of TETyper to cope with high-copy-number TEs and to track specific short-term evolutionary changes, we also applied it to the insertion sequence IS26 within a defined K. pneumoniae outbreak. TETyper is implemented in python and is freely available at https://github.com/aesheppard/TETyper.

Background: The increasing prevalence of nosocomial carbapenemase-producing Enterobacteriaceae is a concern. However, the role of the environment in multispecies outbreaks remains poorly understood. There is increasing recognition that hospital wastewater plumbing may play a role., Methods: Covers were installed on all hoppers (a "toilet-like" waste disposal system) in adult intensive care units (ICUs) of a university hospital; additionally in the surgical ICU, sink trap heating and vibration devices were also installed. Patient acquisitions of Klebsiella pneumoniae carbapenemase-producing organisms (KPCOs) for patients who were admitted to an intervention unit were compared for 18-month preintervention and intervention periods., Results: Sixty hopper covers and 23 sink trap devices were installed. Fifty-six new multispecies KPCO acquisitions occurred preintervention compared to 30 during the intervention. Decreases for all KPCO acquisitions (odds ratio [OR], 0.51; 95% confidence interval [CI], 0.31-0.81; P = .003) and KPCO-positive clinical cultures (OR, 0.29; 95% CI, 0.17-0.48; P < .001) per admission in patients exposed to an intervention unit were observed. The incidence rate ratio was 0.51-fold (95% CI, 0.43-0.61) lower for all KPCO acquisitions during the intervention. The effect of the sink trap devices alone could not be determined, although the proportion of sink drain cultures positive for KPCO decreased (12/15 [80%] sites sampled preintervention vs 40/840 [5%] sampled during the intervention; P = .001)., Conclusions: An intervention targeting wastewater plumbing fixtures, by installation of hopper covers, demonstrated a decrease in patient KPCO acquisitions. Considering wastewater reservoirs in nosocomial transmission of multispecies carbapenemase-producing Enterobacteriaceae may be critical.