Clinical Impact of Plasma Metagenomic Next-Generation Sequencing on Infection Diagnosis and Antimicrobial Therapy in Immunocompromised Patients.

BACKGROUND: Clinical impact of plasma metagenomic next-generation sequencing (mNGS) on infection diagnosis and antimicrobial therapy in immunocompromised patients with suspected infection remains unclear. METHODS: Between March and December 2022, 424 cases with fever, infection history, mechanical ventilation, or imaging abnormalities underwent plasma mNGS testing at a single center. Eleven patients have received solid organ transplantation, and the remaining patients were categorised into febrile neutropenia (FN), non-neutropenia (NN), and non-haematologic disease (NTHD) groups based on immunosuppression severity. The diagnostic rate of infection and the utilisation of antimicrobial agents based on mNGS were assessed. RESULTS: The use of mNGS significantly improved the diagnostic rates for fungi in the FN (56.1%, P = 0.003) and NN (58.8%, P = 0.008) groups versus the NHD group (33.3%). Positive impacts associated with therapy were significantly greater than negative impacts across all three groups (all P < 0.001), and the utilisation of escalation therapy was significantly more frequent in the FN group than in the NN groups (P = 0.006). Over 70% of cases with negative mNGS results across the three groups underwent de-escalation therapy, with >1/3 being discontinued, preventing antimicrobial overuse. CONCLUSIONS: Plasma mNGS has a clinically confirmed positive impact in immunocompromised patients with neutropenia, improving the diagnosis of fungal infections and antimicrobial therapy.

Global genetic diversity and Asian clades evolution: a phylogeographic study of Staphylococcus aureus sequence type 5.

Staphylococcus aureus sequence type (ST) 5 has spread worldwide; however, phylogeographic studies on the evolution of global phylogenetic and Asian clades of ST5 are lacking. This study included 368 ST5 genome sequences, including 111 newly generated sequences. Primary phylogenetic analysis suggested that there are five clades, and geographical clustering of ST5 methicillin-resistant S. aureus (MRSA) was linked to the acquisition of S. aureus pathogenicity islands (SaPIs; enterotoxin gene island) and integration of the prophage φSa3. The most recent common ancestor of global S. aureus ST5 dates back to the mid-1940s, coinciding with the clinical introduction of penicillin. Bayesian phylogeographic inference allowed to ancestrally trace the Asian ST5 MRSA clade to Japan, which may have spread to major cities in China and Korea in the 1990s. Based on a pan-genome-wide association study, the emergence of Asian ST5 clades was attributed to the gain of prophages, SaPIs, and plasmids, as well as the coevolution of resistance genes. Clade IV displayed greater genomic diversity than the Asian MRSA clades. Collectively, our study provides in-depth insights into the global evolution of S. aureus ST5 mainly in China and the United States and reveals that different S. aureus ST5 clades have arisen independently in different parts of the world, with limited geographic dispersal across continents.

Identification of multiple transfer units and novel subtypes of tmexCD-toprJ gene clusters in clinical carbapenem-resistant Enterobacter cloacae and Klebsiella oxytoca.

OBJECTIVES: Tigecycline is a last-resort antibiotic used to treat lethal infections caused by carbapenem-resistant Enterobacterales; however, plasmid-borne tigecycline resistance tmexCD-toprJ gene clusters can confer tigecycline resistance. The aim of the study was to identify novel subtypes and the spread of tmexCD-toprJ. METHODS: Five non-duplicate isolates of different species, carrying tmexCD-toprJ gene clusters or novel subtypes, were isolated from patients across China between November 2018 and June 2019. WGS was performed using Illumina and Nanopore platforms. A phylogenetic tree was constructed using a dataset of 77 sequences carrying the tmexCD-toprJ gene clusters, 72 of which were downloaded from NCBI with a blastn identity cut-off of 95%. RESULTS: We detected six different transfer units and two novel subtypes (tmexC1D1.2-toprJ1 and tmexC2D2.2-toprJ2) of the tmexCD-toprJ gene clusters. Among the six transfer units, three were mediated by IS26, while the rest were presumably mediated by Tn5393, hypothetical integrases (xerD-hp clusters-umuC-integrases-tnfxB2-tmexC2D2-toprJ2-umuC) and hypothetical units (hp-hp-hp-tnfxB2-tmexC2D2.2-toprJ2-ΔTn5393-Tn6292). Moreover, two tmexCD-toprJ-like gene clusters co-located on the same plasmid with blaNDM in five isolates. Phylogenetic analysis revealed that tmexCD-toprJ gene clusters may have originated in Pseudomonas spp., being mainly distributed in Pseudomonas spp. and Klebsiella spp. (64/77). Most tmexCD-toprJ gene clusters in Enterobacterales were located on plasmids, indicating that the gene clusters have a high inter-species transfer risk after transfer to Enterobacterales. CONCLUSIONS: In summary, to the best of our knowledge, this is the first report of tmexCD-toprJ gene clusters being isolated from Enterobacter cloacae and Klebsiella oxytoca, revealing that these multiple transfer units should be further studied because of their clinical significance.

Euxanthone inhibits lipopolysaccharide-induced injury, inflammatory response, and MUC5AC hypersecretion in human airway epithelial cells by the TLR4/MyD88 pathway.

Asthma progression is involved in airway epithelial dysfunction, airway inflammatory response, and mucus hypersecretion. Euxanthone has been found to exhibit cytotoxic activity on several human diseases, such as neurological disorders and cancers. Our study aimed to explore the influence of euxanthone on lipopolysaccharide (LPS)-induced injury, inflammatory response, and mucin 5AC (MUC5AC) hypersecretion in human airway epithelial cells (AECs). Network pharmacology analysis was carried out to analyze the drug targets and key pathways of euxanthone against asthma. Cell injury was evaluated by CCK-8, Lactate dehydrogenase (LDH) release assay, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The production of interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and MUC5AC was measured using enzyme-linked immunosorbent assay (ELISA). MUC5AC mRNA expression was detected by qRT-PCR. Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) protein expression was examined by western blot analysis. Venn diagram showed 14 overlapping targets between euxanthone and asthma. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, we focused on TLR signaling pathway. LPS exposure evoked viability reduction, increased LDH release and apoptosis, and induced production of inflammatory cytokines (IL-6, IL-8, and MCP-1) and MUC5AC hypersecretion in human AECs, which were alleviated by euxanthone. Mechanistically, we validated that euxanthone attenuated LPS-induced activation of TLR4/MyD88 pathway in AECs. Moreover, inhibition of the TLR4/MyD88 pathway enhanced the inhibitory effect of euxanthone on LPS-induced cell injury, inflammatory response and MUC5AC expression. In conclusion, euxanthone attenuated LPS-induced cell injury, inflammatory response, and MUC5AC expression in AECs by inhibiting the activation of TLR4/MyD88 pathway.

Association of dyslipidemia with the severity and mortality of coronavirus disease 2019 (COVID-19): a meta-analysis.

BACKGROUND: The numbers of confirmed cases of coronavirus disease 2019 (COVID-19) and COVID-19 related deaths are still increasing, so it is very important to determine the risk factors of COVID-19. Dyslipidemia is a common complication in patients with COVID-19, but the association of dyslipidemia with the severity and mortality of COVID-19 is still unclear. The aim of this study is to analyze the potential association of dyslipidemia with the severity and mortality of COVID-19. METHODS: We searched the PubMed, Embase, MEDLINE, and Cochrane Library databases for all relevant studies up to August 24, 2020. All the articles published were retrieved without language restriction. All analysis was performed using Stata 13.1 software and Mantel-Haenszel formula with fixed effects models was used to compare the differences between studies. The Newcastle Ottawa scale was used to assess the quality of the included studies. RESULTS: Twenty-eight studies involving 12,995 COVID-19 patients were included in the meta-analysis, which was consisted of 26 cohort studies and 2 case-control studies. Dyslipidemia was associated with the severity of COVID-19 (odds ratio [OR] = 1.27, 95% confidence interval [CI] 1.11-1.44, P = 0.038, I2 = 39.8%). Further, patients with dyslipidemia had a 2.13-fold increased risk of death compared to patients without dyslipidemia (95% CI 1.84-2.47, P = 0.001, I2 = 66.4%). CONCLUSIONS: The results proved that dyslipidemia is associated with increased severity and mortality of COVID-19. Therefore, we should monitor blood lipids and administer active treatments in COVID-19 patients with dyslipidemia to reduce the severity and mortality.

Clinical and microbiological characteristics of adults with hospital-acquired pneumonia: a 10-year prospective observational study in China.

Hospital-acquired pneumonia (HAP) is a significant nosocomial infection; data on the distribution and antimicrobial resistance profiles of HAP in China are limited. We included 2827 adult patients with HAP from the Chinese Antimicrobial Resistance Surveillance of Nosocomial Infections network admitted in 15 Chinese teaching hospitals between 2007 and 2016. Clinical data and antimicrobial susceptibility of isolated pathogens were obtained from the medical records and central laboratory, respectively. Multivariable logistic regression was performed to determine the risk factors for mortality and multidrug resistance (MDR). A total of 386 (13.7%) patients died in the hospital, while 1181 (41.8%) developed ventilator-associated pneumonia (VAP). Active immunosuppressant therapy (OR 1.915 (95% CI 1.475-2.487)), solid tumor (OR 1.860 (95% CI 1.410-2.452)), coma (OR 1.783 (95% CI 1.364-2.333)), clinical pulmonary infection score ≥7 (OR 1.743 (95% CI 1.373-2.212)), intensive care unit stay (OR 1.652 (95% CI 1.292-2.111)), age ≥65 years (OR 1.621 (95% CI 1.282-2.049)), and tracheal cannula insertion (OR 1.613 (95% CI 1.169-2.224)) were independent risk factors for in-hospital mortality. Liver cirrhosis (OR 3.120 (95% CI 1.436-6.780)) and six other variables were independent predictors of MDR. Acinetobacter baumannii (25.6%), Pseudomonas aeruginosa (20.1%), Klebsiella pneumoniae (15.4%), and Staphylococcus aureus (12.6%) were the most common pathogens (MDR prevalence 64.9%). Isolates from VAP patients showed more A. baumannii and less K. pneumoniae and E. coli strains (p < 0.001, respectively) than those from patients without VAP. The proportion of methicillin-resistant S. aureus strains decreased; that of carbapenem-resistant A. baumannii and Enterobacterales strains increased. There had been changes in the antibiotic resistance profiles of HAP pathogens in China. Risk factors for mortality and MDR are important for the selection of antimicrobials for HAP in China.

Whole-Genome Analysis of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Sequence Type 398 Strains Isolated From Patients With Bacteremia in China.

Sequence type (ST) 398 is the most prevalent clone of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). To evaluate the molecular characteristics and phylogeny of Chinese ST398 isolates, 4 MRSA ST398 strains and 4 methicillin-susceptible S. aureus (MSSA) ST398 strains were collected from patients with bacteremia at 6 teaching hospitals in China between 1999 and 2016. Moreover, 689 ST398 genome sequences were downloaded from the GenBank database for comparison. The 4 MRSA ST398 strains were resistant to ß-lactam antibiotics, and 2 strains were also resistant to erythromycin. Among the 4 MSSA ST398 strains, 2 strains displayed multidrug resistance (MDR) and were resistant to penicillin, erythromycin, tetracycline, and gentamicin. The accessory genome of MSSA ST398 was more diverse than that of MRSA ST398. All 4 MRSA ST398 strains carried type V staphylococcal cassette chromosome mec elements; however, MSSA ST398 carried more resistance genes than MRSA ST398. These 4 MRSA ST398 strains carried hemolysin, along with virulence genes associated with immune invasion and protease. Phylogenic analysis showed that the 4 MRSA ST398 strains clustered in 1 clade. The global ST398 phylogeny showed that ST398 was divided into an animal clade and a human clade, and the ST398 strains of this study clustered in the human clade. A small number of human strains were also present in the animal clade and vice versa, suggesting transmission of ST398 between animals and humans. In conclusion, livestock-associated MRSA ST398 has caused severe infections in Chinese hospitals, and it should therefore be paid more attention to and monitored.

Clinical Utility of In-house Metagenomic Next-generation Sequencing for the Diagnosis of Lower Respiratory Tract Infections and Analysis of the Host Immune Response.

BACKGROUND: Only few pathogens that cause lower respiratory tract infections (LRTIs) can be identified due to limitations of traditional microbiological methods and the complexity of the oropharyngeal normal flora. Metagenomic next-generation sequencing (mNGS) has the potential to solve this problem. METHODS: This prospective observational study sequentially enrolled 93 patients with LRTI and 69 patients without LRTI who visited Peking University People's Hospital in 2019. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected using mNGS (DNA and RNA) and traditional microbiological assays. Human transcriptomes were compared between LRTI and non-LRTI, bacterial and viral LRTI, and tuberculosis and nontuberculosis groups. RESULTS: Among 93 patients with LRTI, 20%, 35%, and 65% of cases were detected as definite or probable pathogens by culture, all microbiological tests, and mNGS, respectively. Our in-house BALF mNGS platform had an approximately 2-working-day turnaround time and detected more viruses and fungi than the other methods. Taking the composite reference standard as a gold standard, it had a sensitivity of 66.7%, specificity of 75.4%, positive-predictive value of 78.5%, and negative-predictive value of 62.7%. LRTI-, viral LRTI-, and tuberculosis-related differentially expressed genes were respectively related to immunity responses to infection, viral transcription and response to interferon-γ pathways, and perforin 1 and T-cell receptor B variable 9. CONCLUSIONS: Metagenomic DNA and RNA-seq can identify a wide range of LRTI pathogens, with improved sensitivity for viruses and fungi. Our in-host platform is likely feasible in the clinic. Host transcriptome data are expected to be useful for the diagnosis of LRTIs.

Faecalibacterium prausnitzii produces butyrate to decrease c-Myc-related metabolism and Th17 differentiation by inhibiting histone deacetylase 3.

Decreased levels of Faecalibacterium prausnitzii (F. prausnitzii), whose supernatant plays an anti-inflammatory effect, are frequently found in inflammatory bowel disease (IBD) patients. However, the anti-inflammatory products in F. prausnitzii supernatant and the mechanism have not been fully investigated. Here we found that F. prausnitzii and F. prausnitzii-derived butyrate were decreased in the intestines of IBD patients. Supplementation with F. prausnitzii supernatant and butyrate could ameliorate colitis in an animal model. Butyrate, but not other substances produced by F. prausnitzii, exerted an anti-inflammatory effect by inhibiting the differentiation of T helper 17 (Th17) cells. The mechanism underlying the anti-inflammatory effects of the butyrate produced by F. prausnitzii involved the enhancement of the acetylation-promoted degradation of c-Myc through histone deacetylase 3 (HDAC3) inhibition. In conclusion, F. prausnitzii produced butyrate to decrease Th17 differentiation and attenuate colitis through inhibiting HDAC3 and c-Myc-related metabolism in T cells. The use of F. prausnitzii may be an effective new approach to decrease the level of Th17 cells in the treatment of inflammatory diseases.

Molecular characteristics of oxazolidinone resistance in enterococci from a multicenter study in China.

BACKGROUND: Linezolid-resistant enterococci pose great challenges in clinical practice. The aim of this study is to study the mechanisms underlying the resistance and genetic environment of antimicrobial resistance gene of linezolid-resistant enterococci. RESULTS: The linezolid MICs of 16 enterococci were 4 mg/L to 16 mg/L. Four strains belonged to multi-drug resistant (MDR) bacteria. The sequence types (STs) of 13 enterococci strains performed WGS were diverse: 3 ST476, 1 ST86, ST116, ST480, ST59, ST416, ST21, ST67, ST16, ST585 and ST18. None of them carried multi-drug resistance gene cfr. Only one strain had the G2658 T mutation of target 23S rRNA gene. Thirteen (13/16, 81.3%) strains harbored the novel oxazolidinone resistance gene optrA. WGS analysis showed that the optrA gene was flanked by sequence IS1216E insertion in 13 strains, and optrA was adjacent to transposons Tn558 in two strains and Tn554 in one strain. The optrA gene was identified to be co-localized with fexA, the resistance genes mediated florfenicol resistance in 13 strains, and ermA1, the resistance genes mediated erythromycin resistance in 9 strains, indicating that linezolid-resistant strains may be selected due to non-oxazolidinone antibiotics (i.e. macrolides and florfenicol) usage. CONCLUSION: Our findings demonstrate the high diversity of optrA-carrying genetic platforms. The mobile genetic elements (MGEs) may play an important role in the dissemination of optrA into the enterococci isolates of human origin. The genetic evidence of transferable feature and co-selection of optrA should be gave more attention in clinical practice.

Low levels of pyruvate induced by a positive feedback loop protects cholangiocarcinoma cells from apoptosis.

BACKGROUND: Cancer cells avidly consume glucose and convert it to lactate, resulting in a low pyruvate level. This phenomenon is known as the Warburg effect, and is important for cell proliferation. Although cMyc has often been described as an oncoprotein that preferentially contributes to the Warburg effect and tumor proliferation, mechanisms of action remain unclear. Histone deacetylase 3 (HDAC3) regulates gene expression by removing acetyl groups from lysine residues, as well as has an oncogenic role in apoptosis and contributes to the proliferation of many cancer cells including cholangiocarcinoma (CCA). HDAC inhibitors display antitumor activity in many cancer cell lines. Cancer cells maintain low levels of pyruvate to prevent inhibition of HDAC but the mechanisms remain elusive. The purpose of our study was to explore the role of cMyc in regulating pyruvate metabolism, as well as to investigate whether the inhibitory effect of pyruvate on HDAC3 could hold promise in the treatment of cancer cells. METHODS: We studied pyruvate levels in CCA cell lines using metabolite analysis, and analyzed the relationship of pyruvate levels and cell proliferation with cell viability analysis. We cultivated CCA cell lines with high or low levels of pyruvate, and then analyzed the protein levels of HDAC3 and apoptotic markers via Western Blotting. We then explored the reasons of low levels of pyruvate by using seahorse analysis and 13C6 metabolites tracing analysis, and then confirmed the results using patient tissue protein samples through Western Blotting. Bioinformatics analysis and transfection assay were used to confirm the upstream target of the low levels of pyruvate status in CCA. The regulation of cMyc by HDAC3 was studied through immunoprecipitation and Western Blotting. RESULTS: We confirmed downregulated pyruvate levels in CCA, and defined that high pyruvate levels correlated with reduced cell proliferation levels. Downregulated pyruvate levels decreased the inhibition to HDAC3 and consequently protected CCA cells from apoptosis. Synergistically upregulated LDHA, PKM2 levels resulted in low levels of pyruvate, as well as poor patient survival. We also found that low levels of pyruvate contributed to proliferation of CCA cells and confirmed that the upstream target is cMyc. Conversely, high activity of HDAC3 stabilized cMyc protein by preferential deacetylating cMyc at K323 site, which further contributed to the low pyruvate levels. Finally, this creates a positive feedback loop that maintained the low levels of pyruvate and promoted CCA proliferation. CONCLUSIONS: Collectively, our findings identify a role for promoting the low pyruvate levels regulated by c-Myc, and its dynamic acetylation in cancer cell proliferation. These targets, as markers for predicting tumor proliferation in patients undergoing clinical treatments, could pave the way towards personalized therapies.

Correction to: Low levels of pyruvate induced by a positive feedback loop protects cholangiocarcinoma cells from apoptosis.

Following publication of the original article [1], the authors reported an error in.

Epidemiology of Carbapenem-Resistant Enterobacteriaceae Infections: Report from the China CRE Network.

Carbapenem-resistant Enterobacteriaceae (CRE) infection is highly endemic in China, but estimates of the infection burden are lacking. We established the incidence of CRE infection from a multicenter study that covered 25 tertiary hospitals in 14 provinces. CRE cases defined as carbapenem-nonsusceptible Citrobacter freundii, Escherichia coli, Enterobacter cloacae, or Klebsiella pneumoniae infections during January to December 2015 were collected and reviewed from medical records. Antimicrobial susceptibility testing and carbapenemase gene identification were performed. Among 664 CRE cases, most were caused by K. pneumoniae (73.9%), followed by E. coli (16.6%) and E. cloacae (7.1%). The overall CRE infection incidence per 10,000 discharges was 4.0 and differed significantly by region, with the highest in Jiangsu (14.97) and the lowest in Qinghai (0.34). Underlying comorbidities were found in 83.8% of patients; the median patient age was 62 years (range, 45 to 74 years), and 450 (67.8%) patients were male. Lower respiratory tract infections (65.4%) were the most common, followed by urinary tract infection (16.6%), intra-abdominal infection (7.7%), and bacteremia (7.7%). The overall hospital mortality rate was 33.5%. All isolates showed nonsusceptibility to carbapenems and cephalosporins. The susceptibility rate of polymyxin B was >90%. Tigecycline demonstrated a higher susceptibility rate against E. coli than against K. pneumoniae (90.9% versus 40.2%). Of 155 clinical isolates analyzed, 89% produced carbapenemases, with a majority of isolates producing KPC (50%) or NDM (33.5%)-type beta-lactamases among K. pneumoniae and E. coli The incidence of CRE infection in China was 4.0 per 10,000 discharges. The patient-based disease burden in tertiary hospitals in China is severe, suggesting an urgent need to enhance infection control.

Emergence of mcr-1 and carbapenemase genes in hospital sewage water in Beijing, China.

OBJECTIVES: This study identified and characterized mcr-1-positive Enterobacteriaceae (MCRPE) and carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage water. METHODS: Influent and effluent sewage samples were collected from five tertiary hospitals in Beijing in December 2016. Samples were screened for MCRPE and CPE using antibiotic selection media. Results were confirmed by PCR amplification of ß-lactamase and colistin resistance (mcr-1 and mcr-2) genes and by sequencing. Antimicrobial susceptibility testing, MLST, conjugation and plasmid typing and S1-nuclease-PFGE/Southern blotting were performed for all MCRPE and CPE isolates. RESULTS: Nine MCRPE and 12 CPE isolates were obtained. All mcr-1-positive isolates (n = 9) were Escherichia coli and belonged to eight different STs. The blaKPC-2-positive Enterobacteriaceae included Klebsiella pneumoniae (n = 4), Enterobacter cloacae (n = 4) and Citrobacter freundii (n = 1) isolates. Two C. freundii isolates and one E. cloacae isolate harboured the blaNDM-1 gene. MLST analysis revealed distinct genetic relatedness among all ST11 K. pneumoniae but not among any other carbapenemase-producing isolates. Conjugation and plasmid typing confirmed that three MCRPE isolates harboured mcr-1 on the self-transmissible IncX4 plasmid and the blaNDM-1 gene on the IncX3 plasmid. The sizes of the plasmids harbouring mcr-1, blaNDM-1 and blaKPC-2 were ∼33 to ∼240, ∼40 to ∼75 and ∼30 to ∼90 kb, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report of mcr-1-positive E. coli and blaNDM-1-carrying E. cloacae and C. freundii in hospital sewage water. These findings, especially the diversity of MCRPE and K. pneumoniae ST11 that harbour the blaKPC-2 gene, suggest that monitoring and management of hospital sewage water should be enhanced.

A case report of the metagenomic next-generation sequencing for timely diagnosis of a traveler with nonspecific febrile Q fever.

Q fever is a worldwide distribution disease caused by Coxiella burnetii（C. burnetii）, an obligate intracellular, Gram-negative acidophilic bacterium belonging to γ-proteobacterium. Most patients present with acute Q-fever accompanied by atypical flu-like symptoms, with only 1%-5% of cases may develop into persistent and focally infected foci, mainly manifest as endocarditis, osteomyelitis and prosthetic arthritis. In this case, the patient experienced an unexplained and uninterrupted fever up to 39.2 °C for a week, accompanied by chills and headaches, as well as abnormal liver function. The laboratory reported negative results for blood culture and respiratory-associated pathogens, however, the metagenomic next-generation sequencing (mNGS) reported that detection of 20 sequence reads of C. burnetii in the patient's peripheral blood. In addition, the patient had traveled to Sri Lanka, Iraq and Saudi Arabia before illness. In clinical, the treatment regimen was adjusted from empirically intravenous moxifloxacin 400 mg a day for 1 week to continuously oral minocyline 100 mg twice daily for 2 weeks. The patient was in good health without any adverse sequelae during outpatient visitation and the phone calls follow-up. In conclusion, the mNGS does provide an early and timely diagnostic basis for rare and difficult to culture pathogens, which contributes to the success of clinical anti-infection.

Clinical evaluation of cell-free and cellular metagenomic next-generation sequencing of infected body fluids.

INTRODUCTION: Previous studies have evaluated metagenomic next-generation sequencing (mNGS) of cell-free DNA (cfDNA) for pathogen detection in blood and body fluid samples. However, no study has assessed the diagnostic efficacy of mNGS using cellular DNA. OBJECTIVES: This is the first study to systematically evaluate the efficacy of cfDNA and cellular DNA mNGS for pathogen detection. METHODS: A panel of seven microorganisms was used to compare cfDNA and cellular DNA mNGS assays concerning limits of detection (LoD), linearity, robustness to interference, and precision. In total, 248 specimens were collected between December 2020 and December 2021. The medical records of all the patients were reviewed. These specimens were analysed using cfDNA and cellular DNA mNGS assays, and the mNGS results were confirmed using viral qPCR, 16S rRNA, and internal transcribed spacer (ITS) amplicon next-generation sequencing. RESULTS: The LoD of cfDNA and cellular DNA mNGS was 9.3 to 149 genome equivalents (GE)/mL and 27 to 466 colony-forming units (CFU)/mL, respectively. The intra- and inter-assay reproducibility of cfDNA and cellular DNA mNGS was 100%. Clinical evaluation revealed that cfDNA mNGS was good at detecting the virus in blood samples (receiver operating characteristic (ROC) area under the curve (AUC), 0.9814). In contrast, the performance of cellular DNA mNGS was better than that of cfDNA mNGS in high host background samples. Overall, the diagnostic efficacy of cfDNA combined with cellular DNA mNGS (ROC AUC, 0.8583) was higher than that of cfDNA (ROC AUC, 0.8041) or cellular DNA alone (ROC AUC, 0.7545). CONCLUSION: Overall, cfDNA mNGS is good for detecting viruses, and cellular DNA mNGS is suitable for high host background samples. The diagnostic efficacy was higher when cfDNA and cellular DNA mNGS were combined.

The plasma viral communities associate with clinical profiles in a large-scale haematological patients cohort.

BACKGROUND: Haematological patients exhibit immune system abnormalities that make them susceptible to viral infections. Understanding the relationship between the virome in the blood plasma of haematological patients and their clinical characteristic is crucial for disease management. We aimed to explore the presence of viral pathogens and identify close associations between viral infections and various clinical features. RESULTS: A total of 21 DNA viruses and 6 RNA viruses from 12 virus families were identified from 1383 patients. Patients with haematological diseases exhibited significantly higher diversity, prevalence, and co-detection rates of viral pathogens. During fever episodes, pathogen detection was notably higher, with Epstein-Barr virus (EBV) and Mucorales infections being the most probable culprits for fever symptoms in non-haematological patients. The detection rate of torque teno virus (TTV) significantly increases in haematological patients after transplantation and during primary lung infections. Additionally, TTV-positive patients demonstrate significantly higher absolute neutrophil counts, while C-reactive protein and procalcitonin levels are notably lower. Furthermore, TTV, cytomegalovirus, and parvovirus B19 (B19V) were found to be more prevalent in non-neutropenic patients, while non-viral pathogenic infections, such as Gram-negative bacteria and Mucorales, were more common in neutropenic patients. Pegivirus C (HPgV-C) infection often occurred post-transplantation, regardless of neutropenia. Additionally, some viruses such as TTV, B19V, EBV, and HPgV-C showed preferences for age and seasonal infections. CONCLUSIONS: Analysis of the plasma virome revealed the susceptibility of haematological patients to plasma viral infections at specific disease stages, along with the occurrence of mixed infections with non-viral pathogens. Close associations were observed between the plasma virome and various clinical characteristics, as well as clinical detection parameters. Understanding plasma virome aids in auxiliary clinical diagnosis and treatment, enabling early prevention to reduce infection rates in patients and improve their quality of life. Video Abstract.

mleS in Staphylococcus aureus Contributes to Microaerobic Metabolic Activity, Abscess Formation, and Survival in Macrophages.

Staphylococcus aureus is subdivided into lineages termed sequence types (STs), infections of which necessitate the expression of virulence factors and metabolic adaptation to the host niche. Given that mechanisms underlying the dynamic replacement of sequence types in S. aureus populations have yet to be sufficiently determined, we investigated the role of metabolic determinants in epidemic clones. mleS, encoding the NAD+-dependent malolactic enzyme, was found to be carried by the epidemic clones ST59 and ST398, although not by ST239 and ST5. The genomic location of mleS in the metabolism-associated region flanked by the thiol-specific redox system and glycolysis operon implies that it plays significant roles in metabolism and pathogenesis. Mouse skin abscess caused by the BS19-mleS mutant strain (isogenic mleS mutant in an ST59 isolate) was significantly attenuated and associated with reductions in interleukin-6 (IL-6) and lactic acid production. mleS deletion also impaired S. aureus biofilm formation and survival in RAW264.7 cells. The BS19-mleS-mutant was also characterized by reduced ATP and lactic acid production under microaerobic conditions; however, NAD+/NADH levels remained unaffected. mleS is thus identified as an epidemiological marker that plays an important role in the microaerobic metabolism and pathogenesis of epidemic S. aureus clones. IMPORTANCE Given the importance of metabolic adaptation during infection, new insights are required regarding the pathogenesis of S. aureus, particularly for epidemic clones. We accordingly investigated the role of metabolic determinants that are unique to the epidemic clones ST59 and ST398. Our results provide evidence that the NAD+-dependent malolactic enzyme-coding gene mleS is an epidemiological marker that plays an important role in the microaerobic metabolism and pathogenesis of epidemic S. aureus clones.

Unravelling staphylococcal small-colony variants in cardiac implantable electronic device infections: clinical characteristics, management, and genomic insights.

Objectives: Staphylococcal small-colony variants (SCVs) are common in cardiac implantable electronic device (CIED) infections. This is the first retrospective and multi-case study on CIED infections due to staphylococcal SCVs, aiming to provide a theoretical basis for the clinical management of CIED and device-related infections caused by staphylococcal SCVs. Methods: Ninety patients with culture positive CIED infections were enrolled between 2021 and 2022. We compared the demographic and clinical characteristics of patients with and without SCVs and performed genomic studies on SCVs isolates. Results: Compared to patients without SCVs, those with SCVs had a longer primary pacemaker implantation time and were more likely to have a history of device replacement and infection. They showed upregulated inflammatory indicators, especially higher NEUT% (52.6 vs. 26.8%, P = 0.032) and they had longer hospital stays (median 13 vs. 12 days, P = 0.012). Comparative genomics analysis was performed on Staphylococcus epidermidis wild-type and SCVs. Some genes were identified, including aap, genes encoding adhesin, CHAP domain-containing protein, LPXTG cell wall anchor domain-containing protein, and YSIRK-type signal peptide-containing protein. Conclusion: Staphylococcal SCVs affect the clinical characteristics of CIED infections. The process of staphylococcal SCVs adherence, biofilm formation, and interaction with neutrophils play a vital role.

Transcriptome and Proteome of Methicillin-Resistant Staphylococcus aureus Small-Colony Variants Reveal Changed Metabolism and Increased Immune Evasion.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has become a public health crisis. Recently, we isolated small-colony variants (SCVs) of MRSA, which are characterized by slow growth, decreased virulence, increased antibiotic resistance, and immune evasion. In the present study, we provided proteomic and transcriptomic profiles of clinical MRSA sequence type 239 (ST239) normal strains and SCVs and attempted to identify the key genes or pathways closely related to SCV formation and survival. RNAs and proteins were extracted and subjected to RNA sequencing and mass spectrometry, and the transcriptome and proteome were evaluated via bioinformatic analysis. The results were verified by functional assays. In total, 822 differentially expressed genes (DEGs) and 773 differentially expressed proteins (DEPs) were identified; of these, 286 DEGs and DEPs were correlated and subjected to Kyoto Encyclopedia Genes and Genomes analysis. Some pathways were significant, including ABC transporters, ribosome biogenesis, and metabolic pathways such as glycolysis/gluconeogenesis and the citrate cycle (tricarboxylic acid [TCA] cycle). Based on these results, we found that the downregulation of ABC transporters and the TCA cycle pathway resulted in electron transport chain deficiencies and reduced ATP production in SCVs, leading to a dependence on glycolysis and its upregulation. In addition, the upregulation of capsule polysaccharides and the downregulation of surface proteins prevented phagocytosis and reduced the adhesion of host cells, contributing to immune evasion by SCVs. These findings contribute to a better understanding of the mechanisms of SCV formation and survival. IMPORTANCE Small-colony variants (SCVs) of Staphylococcus aureus have drawn increasing research attention. Owing to their slow growth, atypical colony morphology, and unusual metabolic characteristics, SCVs often cause confusion in the laboratory. Furthermore, clinical treatment of SCVs is challenging owing to their antibiotic resistance and immune evasion, leading to persistent and recurrent infections. However, the mechanisms underlying their formation remain unclear. In this study, we isolated SCVs of methicillin-resistant S. aureus and provided transcriptomic and proteomic profiles of normal strains and SCVs. Based on our analysis, glycolysis upregulation and TCA cycle downregulation affected the electron transport chain and energy supply, leading to slower metabolism. Moreover, capsular biosynthesis was increased, while the number of surface proteins decreased, thus promoting immune evasion by SCVs.