High prevalence of polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae and its within-host evolution to resistance among critically ill scenarios.

PURPOSE: We aimed to explore the prevalence and within-host evolution of resistance in polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae (PHR-CRKP) in critically ill patients. METHODS: We performed an epidemiological analysis of consecutive patients with PHR-CRKP from clinical cases. Our study investigated the within-host resistance evolution and its clinical significance during polymyxin exposure. Furthermore, we explored the mechanisms underlying the dynamic evolution of polymyxin resistance at both subpopulation and genetic levels, involved population analysis profile test, time-killing assays, competition experiments, and sanger sequencing. Additionally, comparative genomic analysis was performed on 713 carbapenemase-producing K. pneumoniae strains. RESULTS: We enrolled 109 consecutive patients, and PHR-CRKP was found in 69.7% of patients without previous polymyxin exposure. 38.1% of PHR-CRKP isolates exhibited polymyxin resistance and led to therapeutic failure in critically ill scenarios. An increased frequency of resistant subpopulations was detected during PHR-CRKP evolution, with rapid regrowth of resistant subpopulations under high polymyxin concentrations, and a fitness cost in an antibiotic-free environment. Mechanistic analysis revealed that diverse mgrB insertions and pmrB hypermutations contributed to the dynamic changes in polymyxin susceptibility in dominant resistant subpopulations during PHR evolution, which were validated by comparative genomic analysis. Several deleterious mutations (e.g. pmrBLeu82Arg, pmrBSer85Arg) were firstly detected during PHR-CRKP evolution. Indeed, specific sequence types of K. pneumoniae demonstrated unique deletions and deleterious mutations. CONCLUSIONS: Our study emphasizes the high prevalence of pre-existing heteroresistance in CRKP, which can lead to polymyxin resistance and fatal outcomes. Hence, it is essential to continuously monitor and observe the treatment response to polymyxins in appropriate critically ill scenarios.

Immune effects of PI3K/Akt/HIF-1α-regulated glycolysis in polymorphonuclear neutrophils during sepsis.

BACKGROUND: Effective removal of pathogenic bacteria is key to improving the prognosis of sepsis. Polymorphonuclear neutrophils (PMNs) are the most important components of innate cellular immunity and play vital roles in clearing pathogenic bacteria. However, the metabolic characteristics and immunomodulatory pathways of PMNs during sepsis have not been investigated. In the present study, we explored the immune metabolism characteristics of PMNs and the mechanism by which neutrophilic glycolysis is regulated during sepsis. METHODS: Metabolomics analysis was performed on PMNs isolated from 14 septic patients, 26 patients with acute appendicitis, and 19 healthy volunteers. Transcriptome analysis was performed on the PMNs isolated from the healthy volunteers and the patients with sepsis to assess glycolysis and investigate its mechanism. Lipopolysaccharide (LPS) was used to stimulate the neutrophils isolated from the healthy volunteers at different time intervals to build an LPS-tolerant model. Chemotaxis, phagocytosis, lactate production, oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) were evaluated. RESULTS: Transcriptomics showed significant changes in glycolysis and the mTOR/HIF-1α signaling pathway during sepsis. Metabolomics revealed that the Warburg effect was significantly altered in the patients with sepsis. We discovered that glycolysis regulated PMNs' chemotaxis and phagocytosis functions during sepsis. Lactate dehydrogenase A (LDHA) downregulation was a key factor in the inhibition of glycolysis in PMNs. This study confirmed that the PI3K/Akt-HIF-1α pathway was involved in the LDHA expression level and also influenced PMNs' chemotaxis and phagocytosis functions. CONCLUSIONS: The inhibition of glycolysis contributed to neutrophil immunosuppression during sepsis and might be controlled by PI3K/Akt-HIF-1α pathway-mediated LDHA downregulation. Our study provides a scientific theoretical basis for the management and treatment of patients with sepsis and promotes to identify therapeutic target for the improvement of immune function in sepsis.

Clinical validation of a multiplex droplet digital PCR for diagnosing suspected bloodstream infections in ICU practice: a promising diagnostic tool.

BACKGROUND: Droplet digital PCR (ddPCR) has emerged as a promising tool of pathogen detection in bloodstream infections (BSIs) in critical care medicine. However, different ddPCR platforms have variable sensitivity and specificity for diverse microorganisms at various infection sites. There is still a lack of prospective clinical studies aimed at validating and interpreting the discrepant ddPCR results for diagnosing BSI in intensive care unit (ICU) practice. METHODS: A prospective diagnostic study of multiplex ddPCR panels was conducted in a general ICU from May 21, 2021, to December 22, 2021. Paired blood cultures (BCs) and ddPCRs (2.5 h) were obtained synchronously to detect the 12 most common BSI pathogens and three antimicrobial resistance (AMR) genes. Firstly, ddPCR performance was compared to definite BSI. Secondly, clinical validation of ddPCR was compared to composite clinical diagnosis. Sensitivity, specificity, and positive and negative predictive values were calculated. Thirdly, the positive rate of AMR genes and related analysis was presented. RESULTS: A total of 438 episodes of suspected BSIs occurring in 150 critical patients were enrolled. BC and ddPCR were positive for targeted bacteria in 40 (9.1%) and 180 (41.1%) cases, respectively. There were 280 concordant and 158 discordant. In comparison with BCs, the sensitivity of ddPCR ranged from 58.8 to 86.7% with an aggregate of 72.5% in different species, with corresponding specificity ranging from 73.5 to 92.2% with an aggregate of 63.1%. Furthermore, the rate of ddPCR+/BC- results was 33.6% (147/438) with 87.1% (128 of 147) cases was associated with probable (n = 108) or possible (n = 20) BSIs. When clinically diagnosed BSI was used as true positive, the final sensitivity and specificity of ddPCR increased to 84.9% and 92.5%, respectively. In addition, 40 blaKPC, 3blaNDM, and 38 mecA genes were detected, among which 90.5% were definitely positive for blaKPC. Further, 65.8% specimens were predicted to be mecA-positive in Staphylococcus sp. according to all microbiological analysis. CONCLUSIONS: The multiplexed ddPCR is a flexible and universal platform, which can be used as an add-on complementary to conventional BC. When combined with clinical infection evidence, ddPCR shows potential advantages for rapidly diagnosing suspected BSIs and AMR genes in ICU practice.

Nomogram for prediction of severe community-acquired pneumonia development in diabetic patients: a multicenter study.

BACKGROUND: Diabetic patients with community-acquired pneumonia (CAP) have an increased risk of progressing to severe CAP. It is essential to develop predictive tools at the onset of the disease for early identification and intervention. This study aimed to develop and validate a clinical feature-based nomogram to identify diabetic patients with CAP at risk of developing severe CAP. METHOD: A retrospective cohort study was conducted between January 2019 to December 2020. 1026 patients with CAP admitted in 48 hospitals in Shanghai were enrolled. All included patients were randomly divided into the training and validation samples with a ratio of 7:3. The nomogram for the prediction of severe CAP development was established based on the results of the multivariate logistic regression analysis and other predictors with clinical relevance. The nomogram was then assessed using receiver operating characteristic curves (ROC), calibration curve, and decision curve analysis (DCA). RESULTS: Multivariate analysis showed that chronic kidney dysfunction, malignant tumor, abnormal neutrophil count, abnormal lymphocyte count, decreased serum albumin level, and increased HbA1c level at admission was independently associated with progression to severe CAP in diabetic patients. A nomogram was established based on these above risk factors and other predictors with clinical relevance. The area under the curve (AUC) of the nomogram was 0.87 (95% CI 0.83-0.90) in the training set and 0.84 (95% CI 0.78-0.90). The calibration curve showed excellent agreement between the predicted possibility by the nomogram and the actual observation. The decision curve analysis indicated that the nomogram was applicable with a wide range of threshold probabilities due to the net benefit. CONCLUSION: Our nomogram can be applied to estimate early the probabilities of severe CAP development in diabetic patients with CAP, which has good prediction accuracy and discrimination abilities. Since included biomarkers are common, our findings may be performed well in clinical practice and improve the early management of diabetic patients with CAP.

Increased mortality of acute respiratory distress syndrome was associated with high levels of plasma phenylalanine.

BACKGROUND: There is a dearth of drug therapies available for the treatment of acute respiratory distress syndrome (ARDS). Certain metabolites play a key role in ARDS and could serve as potential targets for developing therapies against this respiratory disorder. The present study was designed to determine such "functional metabolites" in ARDS using metabolomics and in vivo experiments in a mouse model. METHODS: Metabolomic profiles of blood plasma from 42 ARDS patients and 28 healthy controls were captured using Ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) assay. Univariate and multivariate statistical analysis were performed on metabolomic profiles from blood plasma of ARDS patients and healthy controls to screen for "functional metabolites", which were determined by variable importance in projection (VIP) scores and P value. Pathway analysis of all the metabolites was performed. The mouse model of ARDS was established to investigate the role of "functional metabolites" in the lung injury and mortality caused by the respiratory disorder. RESULTS: The metabolomic profiles of patients with ARDS were significantly different from healthy controls, difference was also observed between metabolomic profiles of the non-survivors and the survivors among the ARDS patient pool. Levels of Phenylalanine, D-Phenylalanine and Phenylacetylglutamine were significantly increased in non-survivors compared to the survivors of ARDS. Phenylalanine metabolism was the most notably altered pathway between the non-survivors and survivors of ARDS patients. In vivo animal experiments demonstrated that high levels of Phenylalanine might be associated with the severer lung injury and increased mortality of ARDS. CONCLUSION: Increased mortality of acute respiratory distress syndrome was associated with high levels of plasma Phenylalanine. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR1800015930. Registered 29 April 2018, http://www.chictr.org.cn/edit.aspx?pid=25609&htm=4.

Plasma PTX3, MCP1 and Ang2 are early biomarkers to evaluate the severity of sepsis and septic shock.

Sepsis is associated with significant mortality. Early diagnosis and prognosis of patients with sepsis is still a difficult clinical challenge. In this study, the ability of plasma PTX3 (pentraxin 3), MCP1 (monocyte chemoattractant protein 1) and Ang (angiopoietin)1/2 was investigated to evaluate the severity of sepsis. Blood samples were obtained from 43 patients with sepsis. A total of 33 post-surgery patients with infections and 25 healthy individuals served as controls. The results showed that plasma PTX3, MCP1 and Ang2 significantly increased in patients on the first day of septic shock onset, while sepsis patients had significantly higher Ang2 level, compared with controls. Furthermore, PTX3, MCP1 and Ang2 had high AUROC values in patients with septic shock on the first day of sepsis onset. The findings suggest that PTX3, MCP1 and Ang2 maybe early predictors to evaluate the severity of sepsis and septic shock with the latest Sepsis 3.0 definitions.

Case report: dose-dependent interaction between dexamethasone and voriconazole in severely ill patients with non-Hodgkin's lymphoma being treated for invasive pulmonary aspergillosis.

Background: Voriconazole is primarily metabolized by CYP2C19 and CYP3A4. Drug interactions that affect this pathway can alter its plasma exposures, resulting in untargeted voriconazole concentrations. Case summary: In this case report, we describe the case of a 64-year-old man who was treated for non-Hodgkin's lymphoma with continuous glucocorticoids co-administrated with voriconazole against invasive pulmonary aspergillosis. A decrease in trough concentration (Cmin) of voriconazole was observed and related with co-administration of dexamethasone in the patient carrying the CYP2C19 *1*2 genotype: voriconazole Cmin/dose ratios of 0.018 (0.1 mg L-1/5.7 mg kg-1 day-1), 0.18 (1 mg L-1/5.7 mg kg-1 day-1), and 0.23 (2 mg L-1/8.6 mg kg-1 day-1) at dexamethasone doses of 20, 12.5, and 2.5 mg, respectively. Sub-therapeutic voriconazole Cmin was associated with high- and moderate-dose dexamethasone (20 and 12.5 mg), leading to failure of antifungal treatment. Conclusion: The extent of voriconazole-dexamethasone interaction was determined by the dose of dexamethasone and associated with the CYP2C19 *1*2 genotype. Therapeutic drug monitoring of voriconazole is necessary to avoid clinically relevant interactions for optimal antifungal therapy.

Effectiveness of ß-blockers in improving 28-day mortality in septic shock: insights from subgroup analysis and retrospective observational study.

Background: In recent years, septic shock remains a common fatal disease in the intensive care unit (ICU). After sufficient fluid resuscitation, some patients still experience tachycardia, which may lead to adverse effects on cardiac function. However, the use of ß-blockers in the treatment of septic shock remains controversial. Thus, the purpose of this study is to evaluate the efficacy of ß-blockers in the treatment of patients with septic shock and explore the most appropriate patient subgroups for this treatment. Methods: This retrospective observational study enrolled septic shock patients from the Medical Information Mart for Intensive Care (MIMIC)-IV and used propensity score matching (PSM) to balance some baseline differences between patients with and without ß-blockers treatment. The primary outcome was the 28-day mortality. Length of stay (LOS) in the ICU and hospital, and the degree of support for organs such as circulatory, respiratory and renal systems were also assessed. Subgroup analysis and multivariate logistic regression were performed to determine the relationship between ß-blockers therapy and 28-day mortality in different patient groups. Results: A total of 4,860 septic shock patients were enrolled in this study and 619 pairs were finally matched after PSM. Our analysis revealed that ß-blocker therapy was associated with a significant improvement in 28-day mortality (21.5% vs. 27.1%; P = 0.020) and led to a prolonged LOS in both the ICU and hospital. Subgroup analysis indicated that there was an interaction between cardiovascular diseases and ß-blocker therapy in patients with septic shock. Patients with pre-existing heart disease or atrial arrhythmias were more likely to derive benefits from ß-blocker treatment. Conclusion: We found ß-blockers therapy was effective to improve 28-day mortality in patients with septic shock. Patients in the subgroup with cardiovascular diseases were more likely to benefit from ß-blockers in mortality.

High-performance lung-targeted bio-responsive platform for severe colistin-resistant bacterial pneumonia therapy.

Polymyxins are the last line of defense against multidrug-resistant (MDR) Gram-negative bacterial infections. However, this last resort has been threatened by the emergence of superbugs carrying the mobile colistin resistance gene-1 (mcr-1). Given the high concentration of matrix metalloproteinase 3 (MMP-3) in bacterial pneumonia, limited plasma accumulation of colistin (CST) in the lung, and potential toxicity of ionic silver (Ag+), we designed a feasible clinical transformation platform, an MMP-3 high-performance lung-targeted bio-responsive delivery system, which we named "CST&Ag@CNMS". This system exhibited excellent lung-targeting ability (>80% in lungs), MMP-3 bio-responsive release property (95% release on demand), and synergistic bactericidal activity in vitro (2-4-fold minimum inhibitory concentration reduction). In the mcr-1+ CST-resistant murine pneumonia model, treatment with CST&Ag@CNMS improved survival rates (70% vs. 20%), reduced bacteria burden (2-3 log colony-forming unit [CFU]/g tissue), and considerably mitigated inflammatory response. In this study, CST&Ag@CNMS performed better than the combination of free CST and AgNO3. We also demonstrated the superior biosafety and biodegradability of CST&Ag@CNMS both in vitro and in vivo. These findings indicate the clinical translational potential of CST&Ag@CNMS for the treatment of lung infections caused by CST-resistant bacteria carrying mcr-1.

Shenfu injection ameliorates endotoxemia-associated endothelial dysfunction and organ injury via inhibiting PI3K/Akt-mediated glycolysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Microcirculatory dysfunction is one of the main characteristics of sepsis. Shenfu Injection (SFI) as a traditional Chinese medicine is widely applied in clinical severe conditions. Recent studies have shown that SFI has the ability to ameliorate sepsis-induced inflammation and to improve microcirculation perfusion. AIM OF THE STUDY: This study aims to investigate the underlying mechanism of SFI for ameliorating sepsis-associated endothelial dysfunction and organ injury. MATERIALS AND METHODS: Side-stream dark-field (SDF) imaging was used to monitor the sublingual microcirculation of septic patients treated with or without SFI. Septic mouse model was used to evaluate the effects of SFI in vivo. Metabolomics and transcriptomics were performed on endothelial cells to identify the underlying mechanism for SFI-related protective effect on endothelial cells. RESULTS: SFI effectively abolished the disturbance and loss of sublingual microcirculation in septic patients. Twenty septic shock patients with or without SFI administration were enrolled and the data showed that SFI significantly improved the levels of total vessel density (TVD), perfused vessel density (PVD), microvascular flow index (MFI), and the proportion of perfused vessels (PPV). The administration of SFI significantly decreased the elevated plasma levels of Angiopoietin-2 (Ang2) and Syndecan-1, which are biomarkers indicative of endothelial damage in sepsis patients. In the mouse septic model in vivo, SFI inhibited the upregulation of endothelial adhesion molecules and Ly6G + neutrophil infiltration while restored the expression of VE-Cadherin in the vasculature of the lung, kidney, and liver tissue. Additionally, SFI reduced the plasma levels of Ang2, Monocyte Chemoattractant Protein-1(MCP1), and Interleukin-6 (IL6), and alleviated liver and kidney injury in septic mice. Moreover, SFI significantly inhibited the inflammatory activation and increased permeability of endothelial cells induced by endotoxins in vitro. By performing metabolomics and transcriptomics, we identified the activation of PI3K/Akt-mediated glycolysis as the underlying mechanism for SFI-related protective effect on endothelial cells. CONCLUSIONS: Our findings revealed that SFI may improve microcirculation perfusion and endothelial function in sepsis via inhibiting PI3K/Akt-mediated glycolysis, providing theoretical evidence for the clinical application of SFI.

Population pharmacokinetics and dosing simulations of meropenem in septic critically ill patients with complicated intra-abdominal infection or pneumonia.

OBJECTIVES: Meropenem pharmacokinetics (PK) may be altered in septic critically ill patients with complicated intra-abdominal infections (cIAI) and pneumonia. We aimed to evaluate the covariates affecting meropenem PK and the performance of different dosing regimens to optimize the PK/pharmacodynamic target. METHODS: Population PK analysis was performed using non-linear mixed-effects modeling. The final model was validated and used to simulate meropenem exposure to assess the probability of attaining the 100%ƒT>MIC target. RESULTS: Forty-six and 14 patients were respectively enrolled for PK analysis and external validation. A one-compartment linear model adequately described the data of 226 concentrations. The typical clearance (CL) and volume of distribution (Vd) were 9.69 L/h and 27.4 L, respectively. Septic shock from cIAI (cIASS) and actual body weight were significant covariates for meropenem Vd in addition to the influential covariates of creatinine clearance (CLCR-CG) and augmented renal clearance for CL. External validation showed the robustness and accuracy of this model. Simulation results proposed continuous infusion (CI) dosing regimens of meropenem against pathogens with MICs ≥ 2 mg/L in patients with cIASS and CLCR-CG ≥ 60 mL/min. CONCLUSIONS: For the patients with cIASS and CLCR-CG ≥ 60 mL/min, CI meropenem is proposed for treatment of less sensitive pathogens with MICs ≥ 2 mg/L.

Effectiveness and safety of Shenfu injection in septic patients with hypoperfusion: A multi-center, open-label, randomized, controlled trial.

Background: To evaluate the effectiveness and safety of the Shenfu injection (SFI) combined with standard bundle treatment in septic patients with hypoperfusion. Method: This study was a multi-center, randomized, open-label, controlled trial conducted in four teaching hospitals in China. The septic patients with hypoperfusion and traditional Chinese medicine (TCM) syndrome with Yang-Qi deficiency were enrolled from January 2019, through September 2020. Eligible patients were randomly allocated in a 1:1 ratio to either receive 60 mL of SFI infusion per day plus standard treatment (SFI group) or standard bundle treatment alone (control group). The primary outcome was 28-day all-cause mortality. Secondary outcomes were 90-day all-cause mortality time to weaning from mechanical ventilation, time to weaning from vasopressors, time to discharge from the ICU and hospital, and laboratory results after randomization. Results: A total of 188 patients completed the trail. This study revealed that the results of the SFI group and the control groups were not statistically significant in 28-day all-cause mortality (10.6% vs. 20.2%, respectively; P=0.106). The infusion of SFI was associated with a significant reduction in the duration of vasopressor use (median=4.0 days, interquartile range [IQR]: 2.0 days-6.0 days vs. median=5.0 days, IQR: 3.0 days-8.0 days, respectively; P=0.043). Patients in the SFI group had statistically greater reductions in plasma lactate levels compared with those in the control group at the first 12 h (median=1.1 mmol/L, IQR: 0.3-2.0 mmol/L vs. median=0.0 mmol/L, IQR: -0.2 to 0.8 mmol/L, respectively; P <0.001) and 24 h (median=1.4 mmol/L, IQR: 0.3-2.2 mmol/L vs. median=0.4 mmol/L, IQR: -0.4 to 1.6 mmol/L, respectively; P=0.001). Conclusion: SFI plus standard therapy did not significantly decrease 28-day all-cause mortality for septic patients with hypoperfusion and TCM syndrome with Yang-Qi deficiency.Trial registration Chinese Clinical Trial Registry Identifier: ChiCTR1800020435.

The ICU-CARB score: a novel clinical scoring system to predict carbapenem-resistant gram-negative bacteria carriage in critically ill patients upon ICU admission.

BACKGROUND: With the widespread spread of carbapenem-resistant gram-negative bacteria (CR-GNB) in medical facilities, the carriage of CR-GNB among critically ill patients has become a significant concern in intensive care units (ICU). This study aimed to develop a scoring system to identify CR-GNB carriers upon ICU admission. METHODS: Consecutive critically ill patients admitted to the ICU of Shanghai Ruijin Hospital between January 2017 and December 2020 were included. The patients were then divided into training and testing datasets at a 7:3 ratio. Parameters associated with CR-GNB carriage were identified using least absolute shrinkage and selection operator regression analysis. Each parameter was assigned a numerical score ranging from 0 to 100 using logistic regression analysis. Subsequently, a four-tier risk-level system was developed based on the cumulative scores, and assessed using the area under the receiver operating characteristic curve (AUC). RESULTS: Of the 1736 patients included in this study, the prevalence of CR-GNB carriage was 10.60%. The clinical scoring system including seven variables (neurological disease, high-risk department history, length of stay ≥ 14 days, ICU history, invasive mechanical ventilation, gastrointestinal tube placement, and carbapenem usage) exhibited promising predictive capabilities. Patients were then stratified using the scoring system, resulting in CR-GNB carriage rates of 2.4%, 12.0%, 36.1%, and 57.9% at the respective risk levels (P < 0.001). Furthermore, the AUC of the developed model in the training set was calculated to be 0.82 (95% CI, 0.78-0.86), while internal validation yielded an AUC of 0.83 (95% CI, 0.77-0.89). CONCLUSIONS: The ICU-CARB Score serves as a straightforward and precise tool that enables prompt evaluation of the risk of CR-GNB carriage at the time of ICU admission, thereby facilitating the timely implementation of targeted pre-emptive isolation.

PKM2/STAT1-mediated PD-L1 upregulation on neutrophils during sepsis promotes neutrophil organ accumulation by serving an anti-apoptotic role.

BACKGROUND: Delayed neutrophil apoptosis during sepsis may impact neutrophil organ accumulation and tissue immune homeostasis. Elucidating the mechanisms underlying neutrophil apoptosis may help identify potential therapeutic targets. Glycolysis is critical to neutrophil activities during sepsis. However, the precise mechanisms through which glycolysis regulates neutrophil physiology remain under-explored, especially those involving the non-metabolic functions of glycolytic enzymes. In the present study, the impact of programmed death ligand-1 (PD-L1) on neutrophil apoptosis was explored. The regulatory effect of the glycolytic enzyme, pyruvate kinase M2 (PKM2), whose role in septic neutrophils remains unaddressed, on neutrophil PD-L1 expression was also explored. METHODS: Peripheral blood neutrophils were isolated from patients with sepsis and healthy controls. PD-L1 and PKM2 levels were determined by flow cytometry and Western blotting, respectively. Dimethyl sulfoxide (DMSO)-differentiated HL-60 cells were stimulated with lipopolysaccharide (LPS) as an in vitro simulation of septic neutrophils. Cell apoptosis was assessed by annexin V/propidium iodide (annexin V/PI) staining, as well as determination of protein levels of cleaved caspase-3 and myeloid cell leukemia-1 (Mcl-1) by Western blotting. An in vivo model of sepsis was constructed by intraperitoneal injection of LPS (5 mg/kg) for 16 h. Pulmonary and hepatic neutrophil infiltration was assessed by flow cytometry or immunohistochemistry. RESULTS: PD-L1 level was elevated on neutrophils under septic conditions. Administration of neutralizing antibodies against PD-L1 partially reversed the inhibitory effect of LPS on neutrophil apoptosis. Neutrophil infiltration into the lung and liver was also reduced in PD-L1-/- mice 16 h after sepsis induction. PKM2 was upregulated in septic neutrophils and promoted neutrophil PD-L1 expression both in vitro and in vivo. In addition, PKM2 nuclear translocation was increased after LPS stimulation, which promoted PD-L1 expression by directly interacting with and activating signal transducer and activator of transcription 1 (STAT1). Inhibition of PKM2 activity or STAT1 activation also led to increased neutrophil apoptosis. CONCLUSION: In this study, a PKM2/STAT1-mediated upregulation of PD-L1 on neutrophils and the anti-apoptotic effect of upregulated PD-L1 on neutrophils during sepsis were identified, which may result in increased pulmonary and hepatic neutrophil accumulation. These findings suggest that PKM2 and PD-L1 could serve as potential therapeutic targets.

SC2sepsis: sepsis single-cell whole gene expression database.

Sepsis, one of the major challenges in the intensive care unit, is characterized by complex host immune status. Improved understandings of the phenotypic changes of immune cells during sepsis and the driving molecular mechanisms are critical to the elucidation of sepsis pathogenesis. Single-cell RNA sequencing (scRNA-seq), which interprets transcriptome at a single-cell resolution, serves as a useful tool to uncover disease-related gene expression signatures of different cell populations in various diseases. It has also been applied to studies on sepsis immunopathological mechanisms. Due to the fact that most sepsis-related studies utilizing scRNA-seq have very small sample sizes and there is a lack of an scRNA-seq database for sepsis, we developed Sepsis Single-cell Whole Gene Expression Database Website (SC2sepsis) (http://www.rjh-sc2sepsis.com/), integrating scRNA-seq datasets of human peripheral blood mononuclear cells from 45 septic patients and 26 healthy controls, with a total amount of 232 226 cells. SC2sepsis is a comprehensive resource database with two major features: (i) retrieval of 1988 differentially expressed genes between pathological and healthy conditions and (ii) automatic cell-type annotation, which is expected to facilitate researchers to gain more insights into the immune dysregulation of sepsis. DATABASE URL: http://www.rjh-sc2sepsis.com/.

Transmission of Nonconjugative Virulence or Resistance Plasmids Mediated by a Self-Transferable IncN3 Plasmid from Carbapenem-Resistant Klebsiella pneumoniae.

Klebsiella pneumoniae poses a critical challenge to clinical and public health. Along with conjugative plasmids, nonconjugative resistance or virulence plasmids associated with carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP), and even carbapenem-resistant and hypervirulent K. pneumoniae (CR-hvKP) strains have been spreading globally. In this study, a clinical CRKP strain KP2648 was isolated, and the transferability of its plasmids was assessed using conjugation experiments. The transconjugants were characterized by polymerase chain reaction (PCR) detection, XbaI and S1-pulsed-field gel electrophoresis (PFGE), and/or whole-genome sequencing. Genetically modified IncN3 plasmids were employed to elucidate the self-transferability and the mobilization mechanisms. KP2648 has three natural plasmids: a nonconjugative IncFIB/IncHI3B virulence plasmid, a nonconjugative IncFII/IncR carbapenem-resistant plasmid, and a self-transferable IncN3 plasmid with a high conjugation frequency (7.54 ± 1.06) × 10-1. The IncN3 plasmid could mobilize the coexisting nonconjugative virulence/resistance plasmids either directly or by employing intermediate E. coli with two forms: a hybrid plasmid fused with IncN3 or a cotransfer with the helper plasmid, IncN3. Various mobile genetic elements, including ISKpn74, ISKpn14, IS26, ISShes11, ISAba11, and Tn3, are involved in the genetic transposition of diverse hybrid plasmids and the cotransfer process during the intra/interspecies transmission. IMPORTANCE Nowadays, the underlying mobilization mechanism and evolutionary processes of nonconjugative virulence or resistance plasmids in Klebsiella pneumoniae remain poorly understood. Our study revealed the high conjugation ability of IncN3 plasmid isolated from carbapenem-resistant K. pneumoniae and confirmed its capability to mobilize the nonconjugative virulence or resistance plasmids. The self-transferable IncN3 plasmid could facilitate the transmission of pathogenicity and genetic evolution of carbapenem-resistant and hypervirulent K. pneumoniae, including hv-CRKP (virulence plasmid obtained by carbapenem-resistant K. pneumoniae) and CR-hvKP (resistance plasmid obtained by hypervirulent K. pneumoniae), warranting further monitoring.

Identifying Drug-Induced Liver Injury Associated With Inflammation-Drug and Drug-Drug Interactions in Pharmacologic Treatments for COVID-19 by Bioinformatics and System Biology Analyses: The Role of Pregnane X Receptor.

Of the patients infected with coronavirus disease 2019 (COVID-19), approximately 14-53% developed liver injury resulting in poor outcomes. Drug-induced liver injury (DILI) is the primary cause of liver injury in COVID-19 patients. In this study, we elucidated liver injury mechanism induced by drugs of pharmacologic treatments against SARS-CoV-2 (DPTS) using bioinformatics and systems biology. Totally, 1209 genes directly related to 216 DPTS (DPTSGs) were genes encoding pharmacokinetics and therapeutic targets of DPTS and enriched in the pathways related to drug metabolism of CYP450s, pregnane X receptor (PXR), and COVID-19 adverse outcome. A network, constructed by 110 candidate targets which were the shared part of DPTSGs and 445 DILI targets, identified 49 key targets and four Molecular Complex Detection clusters. Enrichment results revealed that the 4 clusters were related to inflammatory responses, CYP450s regulated by PXR, NRF2-regualted oxidative stress, and HLA-related adaptive immunity respectively. In cluster 1, IL6, IL1B, TNF, and CCL2 of the top ten key targets were enriched in COVID-19 adverse outcomes pathway, indicating the exacerbation of COVID-19 inflammation on DILI. PXR-CYP3A4 expression of cluster 2 caused DILI through inflammation-drug interaction and drug-drug interactions among pharmaco-immunomodulatory agents, including tocilizumab, glucocorticoids (dexamethasone, methylprednisolone, and hydrocortisone), and ritonavir. NRF2 of cluster 3 and HLA targets of cluster four promoted DILI, being related to ritonavir/glucocorticoids and clavulanate/vancomycin. This study showed the pivotal role of PXR associated with inflammation-drug and drug-drug interactions on DILI and highlighted the cautious clinical decision-making for pharmacotherapy to avoid DILI in the treatment of COVID-19 patients.

Development and Validation of a Nomogram for Predicting the Risk of Coronavirus-Associated Acute Respiratory Distress Syndrome: A Retrospective Cohort Study.

Background: Since the outbreak of coronavirus disease (COVID-19) in December 2019 in Wuhan, it has spread rapidly worldwide. We aimed to establish and validate a nomogram that predicts the probability of coronavirus-associated acute respiratory distress syndrome (CARDS). Methods: In this single-centre, retrospective study, 261 patients with COVID-19 were recruited using positive reverse transcription-polymerase chain reaction tests for severe acute respiratory syndrome coronavirus 2 in Tongji Hospital at Huazhong University of Science and Technology (Wuhan, China). These patients were randomly distributed into the training cohort (75%) and the validation cohort (25%). The factors included in the nomogram were determined using univariate and multivariate logistic regression analyses based on the training cohort. The area under the receiver operating characteristic curve (AUC), consistency index (C-index), calibration curve, and decision curve analysis (DCA) were used to evaluate the efficiency of the nomogram in the training and validation cohorts. Results: Independent predictive factors, including fasting plasma glucose, platelet, D-dimer, and cTnI, were determined using the nomogram. In the training cohort, the AUC and concordance index were 0.93. Similarly, in the validation cohort, the nomogram still showed great distinction (AUC: 0.92) and better calibration. The calibration plot also showed a high degree of agreement between the predicted and actual probabilities of CARDS. In addition, the DCA proved that the nomogram was clinically beneficial. Conclusion: Based on the results of laboratory tests, we established a predictive model for acute respiratory distress syndrome in patients with COVID-19. This model shows good performance and can be used clinically to identify CARDS early. Trial Registration: Ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (No.:(2020) Linlun-34th).

A bifunctional electrochemical sensor for simultaneous determination of electroactive and non-electroactive analytes: A universal yet very effective platform serving therapeutic drug monitoring.

A novel electrochemical sensor for therapeutic drug monitoring (TDM) of antibiotic Zavicefta was developed in this work. The main components of Zavicefta are ceftazidime (CFZ) and avibactam (AVI), wherein CFZ can be oxidized directly on electrode surface, while AVI is non-electroactive. In order to realize the simultaneous detection of the two analytes on one sensor, we elaborately constructed a dual-template molecularly imprinted polymer (MIP) modified electrode with CFZ and AVI as the templates, and further developed a dual-signal strategy. During detection, due to the inhibition effect caused by rebinding of MIP to the targets, the peak current of probe ferricyanide ([Fe(CN)6]3-) showed obvious suppression. Meanwhile, a new peak appeared, which originated from the oxidation of CFZ. The proposed sensor presented a wide linear range of 50-1000 µM for CFZ and 1-1000 µM for AVI, with the respective detection limit of 35 µM and 0.5 µM (S/N = 3). Owing to the imprinting effect of MIP, the sensor displayed excellent selectivity and anti-interference ability, which was successfully applied for TDM of CFZ and AVI in human serum and a live rabbit. The results agreed well with those from HPLC. This work is expected to offer a promising avenue for simultaneous measurement of electroactive and non-electroactive substances, and extend the potential of electrochemical sensors in medical field.

Validation and Extrapolation of a Multimodal Infection Prevention and Control Intervention on Carbapenem-Resistant Klebsiella pneumoniae in an Epidemic Region: A Historical Control Quasi-Experimental Study.

Objective: To verify the effects of comprehensive infection prevention and control (IPC) interventions for the prevention of the cross-transmission of carbapenem-resistant Klebsiella pneumoniae (CRKP) within intensive care units (ICUs) in an epidemic region. Methods: A historical control, quasi-experimental design was performed. The study was conducted between January 2017 and December 2019, following the implementation of a multimodal IPC bundle. The baseline period was established from January 2013 to June 2013, when only basic IPC measures were applied. Results: A total of 748 patients were enrolled during the entire study. The incidence of ICU-acquired CRKP colonization/infection was 1.16 per 1,000 patient-days during the intervention period, compared with 10.19 per 1,000 patient-days during the baseline period (p = 0.002). The slope of the monthly incidence of CRKP at admission showed an increasing trend (p = 0.03). The incidence of ICU-acquired catheter-related bloodstream infections caused by CRKP decreased from 2.54 to 0.96 per 1,000 central-line-days (p = 0.08). Compliance with contact precautions and terminal room disinfection improved during the intervention period. All environmental surface culture samples acquired after terminal room disinfection were negative for CRKP. Conclusion: Our findings suggest that in epidemic settings, multimodal IPC intervention strategies and consistent monitoring of compliance, may limit the spread of CRKP in ICUs.