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ABSTRACT Central nervous system infections (CNSIs) are common complications after neurosurgery with a poor prognosis. The traditional microbiological culture methodology has a low detection rate and time consuming. Metagenomic next-generation sequencing (mNGS) has demonstrated the advantages of being faster, more accurate, and more comprehensive in clinical microbiology. Previous studies had suggested that mNGS had a high sensitivity in the diagnosis of CNSIs. Whether the application of mNGS has health economic value in clinical applications remains to be studied. We designed a prospective, single-center, superiority randomized controlled trial to compare the cost-effectiveness of mNGS with traditional methods for diagnosing CNSIs using a decision tree model. A total of 204 patients will be enrolled and randomly assigned to either the mNGS group or the traditional method group. The two groups of patients entered different decision points according to different clinical manifestations and examination results. They will be then given treatment decisions by a panel of specialists at the corresponding decision point. The primary outcome is the incremental cost-effectiveness ratio, which is the increased cost for every 1% increase in recovery rate. The secondary outcomes are a comparison of time cost, detection cost, and costs associated with antibiotics treatment between the two groups.IMPORTANCEDiagnosing and treating postoperative central nervous system infections (PCNSIs) remains challenging due to the low detection rate and time-consuming nature of traditional methods for identifying microorganisms in cerebrospinal fluid. Metagenomic next-generation sequencing (mNGS) technology provides a rapid and comprehensive understanding of microbial composition in PCNSIs by swiftly sequencing and analyzing the microbial genome. The current study aimed to assess the economic impact of using mNGS versus traditional bacterial culture-directed PCNSIs diagnosis and therapy in post-neurosurgical patients from Beijing Tiantan Hospital. mNGS is a relatively expensive test item, and whether it has the corresponding health-economic significance in the clinical application of diagnosing intracranial infection has not been studied clearly. Therefore, the investigators hope to explore the clinical application value of mNGS detection in PCNSIs after neurosurgery.

ABSTRACT H6 influenza A viruses are enzootic and genetically diverse in both domestic poultry and wild birds, occasionally causing spillovers to mammals, including humans, posing threat to public health. However, the antigenic sites of the hemagglutinin (HA) of H6 are poorly understood. In this study, a panel of murine monoclonal antibodies (MAbs) with different reactivity spectra against HA of H6 was generated and nine key residues of antigenic epitopes in H6 were identified through escape mutants of these MAbs. Of these, residues 139 and 140 belonged to antigenic Sa, residue 89 belonged to antigenic Cb, residue 149 belonged to Ca2, while residue 221 belonged to Ca1 regarding the H1 antigenic sites. However, the other four residues 69, 120, 124, and 246 were not located in the identified antigenic regions of H1. Although most HA variants of the escape mutants had the preference for avian-like receptor, whereas escape mutants m3B9 and m4C2 increased the binding affinity for both avian and human-like receptors, and m1A5 and m6D11 decreased the binding affinity for avian-like receptors. Moreover, a single dose of MAbs 4C2 or 6E3 could fully protect mice against lethal challenge of mouse-adapted A/Eurasian teal/Jiangxi/2018WB0417(H6N2) (MA E-Teal/417) in the prophylactic treatment and also could provide partial protection in the therapeutic treatment. Overall, the generated MAbs and the identified key residues of antigenic epitopes not only provide novel insights into the key markers for monitoring the antigenic variation of H6 but also demonstrate the potential for developing MAb-based passitive treatments for H6. IMPORTANCE Since the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

ABSTRACT Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

ABSTRACT The Infectious Disease Surveillance of Pediatrics (ISPED) program was established in 2015 to monitor and analyze the trends of bacterial epidemiology and antimicrobial resistance (AMR) in children. Clinical bacterial isolates were collected from 11 tertiary care children’s hospitals in China in 2016 to 2020. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer method or automated systems, with interpretation according to the Clinical and Laboratory Standards Institute 2019 breakpoints. A total of 288,377 isolates were collected, and the top 10 predominant bacteria were Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Streptococcus pyogenes, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Acinetobacter baumannii. In 2020, the coronavirus disease 2019 (COVID-19) pandemic year, we observed a significant reduction in the proportion of respiratory tract samples (from 56.9% to 44.0%). A comparable reduction was also seen in the primary bacteria mainly isolated from respiratory tract samples, including S. pneumoniae, H. influenzae, and S. pyogenes. Multidrug-resistant organisms (MDROs) in children were commonly observed and presented higher rates of drug resistance than sensitive strains. The proportions of carbapenem-resistant K. pneumoniae (CRKP), carbapenem-resistant A. baumannii (CRAB), carbapenem-resistant P. aeruginosa (CRPA), and methicillin-resistant S. aureus (MRSA) strains were 19.7%, 46.4%%, 12.8%, and 35.0%, respectively. The proportions of CRKP, CRAB, and CRPA strains all showed decreasing trends between 2015 and 2020. Carbapenem-resistant Enterobacteriaceae (CRE) and CRPA gradually decreased with age, while CRAB showed the opposite trend with age. Both CRE and CRPA pose potential threats to neonates. MDROs show very high levels of AMR and have become an urgent threat to children, suggesting that effective monitoring of AMR and antimicrobial stewardship among children in China are required. IMPORTANCE AMR, especially that involving multidrug-resistant organisms (MDROs), is recognized as a global threat to human health; AMR renders infections increasingly difficult to treat, constituting an enormous economic burden and producing tremendous negative impacts on patient morbidity and mortality rates. There are many surveillance programs in the world to address AMR profiles and MDRO prevalence in humans. However, published studies evaluating the overall AMR rates or MDRO distributions in children are very limited or are of mixed quality. In this study, we showed the bacterial epidemiology and resistance profiles of primary pathogens in Chinese children from 2016 to 2020 for the first time, analyzed MDRO distributions with time and with age, and described MDROs’ potential threats to children, especially low-immunity neonates. Our study will be very useful to guide antiinfection therapy in Chinese children, as well as worldwide pediatric patients.

ABSTRACT The balance in the functions of hemagglutinin (HA) and neuraminidase (NA) plays an important role in influenza virus genesis. However, whether and how N2 neuraminidase-specific antibodies may affect the attributes of HA remains to be investigated. In this study, we examined the presence of amino acid mutations in the HA of mutants selected by incubation with N2-specific monoclonal antibodies (MAbs) and compared the HA properties to those of the wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus. The higher NA inhibition (NI) ability of N2-specific MAbs was found to result in greater proportions of mutations in the HA head. The HA mutations affected the thermal stability, switched the binding preferences from α2,6-linked sialic acid receptor to α2,3-linked sialic acid receptor, and promoted viral growth in mouse lungs. These mutations also caused significant HA antigenic drift as they decreased hemagglutination inhibition (HI) titers. The evolutionary analysis also proved that some HA mutations were highly correlated with NA antibody pressure. Our data demonstrate that HA mutations caused by NA-specific antibodies affect HA properties and may contribute to HA evolution. IMPORTANCE HA binds with the sialic acid receptor on the host cell and initiates the infection mode of influenza virus. NA cleaves the connection between receptor and HA of newborn virus at the end of viral production. The HA-NA functional balance is crucial for viral production and interspecies transmission. Here, we identified mutations in the HA head of H9N2 virus caused by NA antibody pressure. These HA mutations changed the thermal stability and switched the receptor-binding preference of the mutant virus. The HI results indicated that these mutations resulted in significant antigenic drift in mutant HA. The evolutionary analysis also shows that some mutations in HA of H9N2 virus may be caused by NA antibody pressure and may correlate with the increase in H9N2 infections in humans. Our results provide new evidence for HA-NA balance and an effect of NA antibody pressure on HA evolution.

ABSTRACT Dolosigranulum pigrum is positively associated with indicators of health in multiple epidemiological studies of human nasal microbiota. Knowledge of the basic biology of D. pigrum is a prerequisite for evaluating its potential for future therapeutic use; however, such data are very limited. To gain insight into D. pigrum’s chromosomal structure, pangenome, and genomic stability, we compared the genomes of 28 D. pigrum strains that were collected across 20 years. Phylogenomic analysis showed closely related strains circulating over this period and closure of 19 genomes revealed highly conserved chromosomal synteny. Gene clusters involved in the mobilome and in defense against mobile genetic elements (MGEs) were enriched in the accessory genome versus the core genome. A systematic analysis for MGEs identified the first candidate D. pigrum prophage and insertion sequence. A systematic analysis for genetic elements that limit the spread of MGEs, including restriction modification (RM), CRISPR-Cas, and deity-named defense systems, revealed strain-level diversity in host defense systems that localized to specific genomic sites, including one RM system hot spot. Analysis of CRISPR spacers pointed to a wealth of MGEs against which D. pigrum defends itself. These results reveal a role for horizontal gene transfer and mobile genetic elements in strain diversification while highlighting that in D. pigrum this occurs within the context of a highly stable chromosomal organization protected by a variety of defense mechanisms. IMPORTANCE Dolosigranulum pigrum is a candidate beneficial bacterium with potential for future therapeutic use. This is based on its positive associations with characteristics of health in multiple studies of human nasal microbiota across the span of human life. For example, high levels of D. pigrum nasal colonization in adults predicts the absence of Staphylococcus aureus nasal colonization. Also, D. pigrum nasal colonization in young children is associated with healthy control groups in studies of middle ear infections. Our analysis of 28 genomes revealed a remarkable stability of D. pigrum strains colonizing people in the United States across a 20-year span. We subsequently identified factors that can influence this stability, including genomic stability, phage predators, the role of MGEs in strain-level variation, and defenses against MGEs. Finally, these D. pigrum strains also lacked predicted virulence factors. Overall, these findings add additional support to the potential for D. pigrum as a therapeutic bacterium.

Oyster mushrooms are grown commercially worldwide, especially in many developing countries, for their easy cultivation and high biological efficiency. Pleurotus cornucopiae is one of the main oyster mushroom species because of its gastronomic value and nutraceutical properties. Cap color is an important trait, since consumers prefer dark mushrooms, which are now represented by only a small portion of the commercial varieties. Breeding efforts are required to improve quality- related traits to satisfy various demands of consumers. Here, we present a saturated genetic linkage map of P. cornucopiae constructed by using a segregating population of 122 monokaryons and 3,449 single nucleotide polymorphism (SNP) markers generated by the 2b-RAD approach. The map contains 11 linkage groups covering 961.6 centimorgans (cM), with an average marker spacing of 0.27 cM. The genome of P. cornucopiae was de novo sequenced, resulting in 425 scaffolds (>1,000 bp) with a total genome size of 35.1 Mb. The scaffolds were assembled to the pseudochromosome level with the assistance of the genetic linkage map. A total of 97% SNP markers (3,357) were physically localized on 140 scaffolds that were assigned to 11 pseudochromosomes, with a total of 32.5 Mb, representing 92.5% of the whole genome. Six quantitative trait loci (QTL) controlling cap color of P. cornucopiae were detected, accounting for a total phenotypic variation of 65.6%, with the highest value for the QTL on pseudochromosome 5 (18%). The results of our study provide a solid base for marker-assisted breeding for agronomic traits and especially for studies on biological mechanisms controlling cap color in oyster mushrooms. IMPORTANCE Oyster mushrooms are produced and consumed all over the world. Pleurotus cornucopiae is one of the main oyster mushroom species. Dark-cap oyster mushrooms are becoming more and more popular with consumers, but dark varieties are rare on the market. Prerequisites for efficient breeding programs are the availability of high-quality whole genomes and genetic linkage maps. Genetic studies to fulfill some of these prerequisites have hardly been done for P. cornucopiae. In this study, we de novo sequenced the genome and constructed a saturated genetic linkage map for P. cornucopiae. The genetic linkage map was effectively used to assist the genome assembly and identify QTL that genetically control the trait cap color. As well, the genome characteristics of P. cornucopiae were compared to the closely related species Pleurotus ostreatus. The results provided a basis for understanding the genetic background and marker-assisted breeding of this economically important mushroom species. [ABSTRACT FROM AUTHOR]

Oyster mushrooms have a high biological efficiency and are easy to cultivate, which is why they are produced all over the world. Cap color is an important commercial trait for oyster mushrooms. Little is known about the genetic mechanism of the cap color trait in oyster mushrooms, which limits molecular breeding for the improvement of cap color-type cultivars. In this study, a 0.8-Mb major quantitative trait locus (QTL) region controlling cap color in the oyster mushroom Pleurotus cornucopiae was mapped on chromosome 7 through bulked-segregant analysis sequencing (BSA-seq) and extreme-phenotype genome-wide association studies (XP-GWAS). Candidate genes were further selected by comparative transcriptome analysis, and a tyrosinase gene (PcTYR) was identified as the highest-confidence candidate gene. Overexpression of PcTYR resulted in a significantly darker cap color, while the cap color of RNA interference (RNAi) strains for this gene was significantly lighter than that of the wild-type (WT) strains, suggesting that PcTYR plays an essential role in cap color formation. This is the first report about fine mapping and functional verification of a gene controlling cap color in oyster mushrooms. This will enhance our understanding of the genetic basis for cap color formation in oyster mushrooms and will facilitate molecular breeding for cap color. [ABSTRACT FROM AUTHOR]