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Abstract Background In recent years, the hypervirulent and carbapenem-resistant Klebsiella pneumoniae has been increasingly reported worldwide. The objective of this study was to compare the antibiotic resistance and virulence profiles of carbapenem-resistant hypervirulent K.pneumoniae (CR-hvKP) and hypervirulent carbapenem-resistant K.pneumoniae (hv-CRKP) and identify the prevailing strain in clinical settings. Methods In this study, hv-CRKP or CR-hvKP were identified based on the results of whole-genome analysis (WGS), multilocus sequence typing (MLST) and the antimicrobial susceptibility testing. We then compared antibiotic resistance and virulence profiles between CR-hvKP and hv-CRKP through the antimicrobial susceptibility testing and a series of virulence experiments including biofilm formation ability detection method, the resistance test against human serum, siderophore production test, neutrophil phagocytosis assay and Galleria mellonella infection model. Additionally, pathway enrichment analysis was conducted to assess the effect of SNPs on the phenotype. Results In this study, we categorized 17.4% of hypervirulent and carbapenem-resistant K. pneumoniae strains as CR-hvKP and 82.6% as hv-CRKP. Among them, 84.2% (16/19) of CR-hvKP strains harboring carbapenemase genes exhibited lower imipenem and meropenem MIC values compared to hv-CRKP strains. The virulence potential of hv-CRKP and CR-hvKP was confirmed by using virulence experiments in vitro and in vivo, showing that virulence of the CR-hvKP strains was comparable to that of hv-CRKP strains. Notably, the 90 hv-CRKP strains were classified into 3 different ST types and 8 capsule types, each showing varying degrees of resistance and virulence. We observed that subclonal replacement was within the predominant hv-CRKP clone, with the ST11-KL64 strain, characterized by high-level resistance and virulence emerging as the currently prevailing subclone, replacing ST11-KL47. KEGG enrichment analysis showed that pathways associated with the citrate cycle (TCA cycle), glycolysis/gluconeogenesis, glutathione metabolism, two-component regulatory system, and folate metabolism were significantly enriched among the group expressing different levels of capsular polysaccharides. Conclusions The hv-CRKP strains exhibited a greater survival advantage in the hospital environment than CR-hvKP strains. Notably, the ST11-KL64 hv-CRKP strain which displayed a high level of resistance and hypervirulence, warrants the most clinical vigilance. Clinical trial number Not applicable.

ABSTRACT Staphylococcus aureus is an important human pathogen in both community and hospital settings that often causes persistent and recurrent infections. The continuous emergence of multidrug-resistant strains and the lag in antibiotic development make the treatment of S. aureus more challenging. The biofilm formation of S. aureus is an important reason for persistent infection. In this study, we demonstrated that a small-molecule compound S-342-3 can effectively inhibit the biofilm formation of S. aureus. The crystal violet semi-quantitative assays revealed that at a sub-minimum inhibitory concentration of 4 µg/mL, S-342-3 reduced S. aureus biofilm mass by 57.43%, 52.14%, and 25.49%. Using confocal laser scanning microscopy, we observed that the biofilm was more incompact and less uniform upon the treatment of S-342-3. At a sub-inhibitory concentration (4 µg/mL), the S-342-3 can effectively reduce the production of polysaccharide intercellular adhesin (PIA) and suppress the initial adhesion of cells in the biofilm. Consistently, the results of RT-qPCR revealed that the expression of genes associated with biofilm formation was downregulated by S-342-3 (P < 0.05). However, we found that treatment with S-342-3 resulted in a significant decrease in the expression of global regulatory genes agrA and sarA (P < 0.05), which play a key role in promoting cell surface attachment and PIA production in S. aureus biofilms. Also importantly, we experimentally proved that the S-342-3 was not toxic to A549 alveolar epithelial cells and the Galleria mellonella larvae. Collectively, these results suggest that the S-342-3 may be a promising anti-biofilm drug candidate with excellent biosafety, which can be further explored for its practical application in health care. IMPORTANCE Biofilms are an important virulence factor in Staphylococcus aureus and are characterized by a structured microbial community consisting of bacterial cells and a secreted extracellular polymeric matrix. Inhibition of biofilm formation is an effective measure to control S. aureus infection. Here, we have synthesized a small molecule compound S-342-3, which exhibits potent inhibition of biofilm formation in both MRSA and MSSA. Further investigations revealed that S-342-3 exerts inhibitory effects on biofilm formation by reducing the production of polysaccharide intercellular adhesin and preventing bacterial adhesion. Our study has confirmed that the inhibitory effect of S-342-3 on biofilm is achieved by downregulating the expression of genes responsible for biofilm formation. In addition, S-342-3 is non-toxic to Galleria mellonella larvae and A549 cells. Consequently, this study demonstrates the efficacy of a biologically safe compound S-342-3 in inhibiting biofilm formation in S. aureus, thereby providing a promising antibiofilm agent for further research.

The increasing prevalence of multidrug-resistant (MDR) Gram-negative bacteria and comparatively limited options of antibiotics pose a major threat to public health worldwide. Polymyxin B is the last resort against extensively resistant Gram-negative bacterial infections. However, a large number of Gram-negative bacteria exhibited high-level resistance to Polymyxin B, bringing challenges for antimicrobial chemotherapy. Combination therapies using polymyxins and other antibiotics are recommended to treat multidrug-resistant pathogens. In this study, we selected Gram-negative bacterial strains, including Klebsiella pneumoniae and Escherichia coli, to explore whether fusidic acid and polymyxin B have a synergistic killing effect. Through broth microdilution, we observed that minimum inhibitory concentrations (MICs) against polymyxin B in the isolates tested were significantly reduced by the addition of fusidic acid. Notably, chequerboard analysis indicated a synergistic effect between polymyxin B and fusidic acid. In addition, subsequent time-kill experiments showed that the combination of polymyxin B and fusidic acid was more effective than a single drug in killing bacteria. Finally, our investigation utilizing the murine model revealed a higher survival rate in the combination therapy group compared to the monotherapy group. Our research findings provide evidence of the synergistic effect between polymyxin B and fusidic acid. Fusidic acid was shown to increase the sensitivity of multi-drug resistant E. coli and K. pneumoniae to polymyxin B, thereby enhancing its bactericidal activity. This study provides new insights into a potential strategy for overcoming polymyxin B resistance, however, further investigations are required to evaluate their feasibility in real clinical settings.

Carbapenem-resistant Enterobacterales (CRE) infection is highly endemic in China; Klebsiella pneumoniae carbapenemase (KPC) 2–producing CRE is the most common, whereas KPC-3–producing CRE is rare. We report an outbreak of KPC-3–producing Enterobacterales infection in China. During August 2020–June 2021, 25 blaKPC-3–positive Enterobacteriale isolates were detected from 24 patients in China. Whole-genome sequencing analysis revealed that the blaKPC-3 genes were harbored by IncX8 plasmids. The outbreak involved clonal expansion of KPC-3–producing Serratia marcescens and transmission of blaKPC-3 plasmids across different species. The blaKPC-3 plasmids demonstrated high conjugation frequencies (10−3 to 10−4). A Galleria mellonella infection model showed that 2 sequence type 65 K2 K. pneumoniae strains containing blaKPC-3 plasmids were highly virulent. A ceftazidime/avibactam in vitro selection assay indicated that the KPC-3–producing strains can readily develop resistance. The spread of blaKPC-3–harboring IncX8 plasmids and these KPC-3 strains should be closely monitored in China and globally.

ABSTRACT ST22 MRSA (methicillin-resistant Staphylococcus aureus) strains are only sporadically reported in China. Through the phylogenetic reconstruction of 30 ST22 strains from China and 480 ST22 strains from global sources, we found that the global ST22 strains can be divided into three clades (I, II, and III). The China ST22 strains were found primarily in clade II (IIb and IIc) and also in clade III, indicating that the China ST22-MRSA clones have different origins. The China subclade IIb strains (SCCmec Vb-t309) may evolve from the native ST22 MSSA clone, while the China IIc strains may have spread from other countries. Subclade IIc (SCCmecIVa-t309) strains exhibited particularly strong lethality and invasiveness in Galleria mellonella infection and mouse skin abscess models in comparison to USA300 and other dominant China HA-MRSA (ST5 and ST239) or CA-MRSA (ST59) strains. This study described the emergence of a highly virulent ST22 MRSA subclade and improved our insight into the molecular epidemiology of ST22 strains in China. IMPORTANCE ST22 is a successful hospital-associated MRSA lineage which first appeared in the United Kingdom as EMRSA-15. At present, ST22 MRSA clones are spreading rapidly around the world and even replaced other dominant clones in some regions. We placed the Chinese ST22 in the worldwide phylogeny of ST22, demonstrating a distinctive molecular epidemiology and to our knowledge, this is the first time that a novel clade of ST22 has been found in China. Among the 15 ST22 MRSA strains belonging to the novel clade, 14 ST22 SCCmecIVa strains from different regions carried both pvl and tst and displayed significantly higher in vitro and in vivo virulence in comparison to other clade/subclade ST22 strains as well as other common China HA-MRSA or CA-MRSA strains. The further spread of this subclade of strains could pose a serious threat to the health system in China and other regions.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and restricted therapeutic options pose a global threat to public health. Aminoglycosides are a wise choice, which can effectively reduce the mortality rate when combined with β-lactam drugs. However, in this study, we identified a ST15-KL112 CRKP FK3006 which not only exhibited resistance to carbapenems, but also exhibited high level resistance to aminoglycosides. In addition to the multidrug resistant phenotype, FK3006 also owned typical pathogenic characteristic, including hypermucoviscosity and hypervirulence phenotypes. According to the whole-genome sequencing, one pLVPK-like virulence plasmid, and three key resistant plasmids (blaOXA-232, blaCTX-M-15, and rmtF) were observed in FK3006. Compared to other typical ST15 CRKP, the presence of pLVPK-like virulence plasmid (p3006-2) endowed the FK3006 with high virulence features. High siderophore production, more cell invasive and more resistant to serum killing was observed in FK3006. The Galleria mellonella infection model also further confirmed the hypervirulent phenotype of FK3006 in vivo. Moreover, according to the conjugation assay, p3006-2 virulence plasmid also could be induced transfer with the help of conjugative IncFIIK p3006-11 plasmid (blaCTX-M-15). In addition to the transmissible plasmid, several insertion sequences and transposons were found around blaCTX-M-15, and rmtF to generate the mobile antimicrobial resistance island (ARI), which also make a significant contribution to the dissemination of resistant determinants. Overall, we reported the uncommon co-existence of blaOXA-232, rmtF-encoding plasmids, and pLVPK-like virulence plasmid in ST15-KL112 K. pneumoniae. The dissemination threatens of these high-risk elements in K. pneumoniae indicated that future studies are necessary to evaluate the prevalence of such isolates.

Solanales, an order of flowering plants, contains the most economically important vegetables among all plant orders. To date, many Solanales genomes have been sequenced. However, the evolutionary processes of polyploidization events in Solanales and the impact of polyploidy on species diversity remain poorly understood. We compared two representative Solanales genomes (Solanum lycopersicum L. and Ipomoea triloba L.) and the Vitis vinifera L. genome and confirmed two independent polyploidization events. Solanaceae common hexaploidization (SCH) and Convolvulaceae common hexaploidization (CCH) occurred ∼43–49 and ∼40–46 million years ago (Mya), respectively. Moreover, we identified homologous genes related to polyploidization and speciation and constructed multiple genomic alignments with V. vinifera genome, providing a genomic homology framework for future Solanales research. Notably, the three polyploidization-produced subgenomes in both S. lycopersicum and I. triloba showed significant genomic fractionation bias, suggesting the allohexaploid nature of the SCH and CCH events. However, we found that the higher genomic fractionation bias of polyploidization-produced subgenomes in Solanaceae was likely responsible for their more abundant species diversity than that in Convolvulaceae. Furthermore, through genomic fractionation and chromosomal structural variation comparisons, we revealed the allohexaploid natures of SCH and CCH, both of which were formed by two-step duplications. In addition, we found that the second step of two paleohexaploidization events promoted the expansion and diversity of β-amylase (BMY) genes in Solanales. These current efforts provide a solid foundation for future genomic and functional exploration of Solanales.

Abstract Background Duplicated gene pairs produced by ancient polyploidy maintain high sequence similarity over a long period of time and may result from illegitimate recombination between homeologous chromosomes. The genomes of Asian cultivated rice Oryza sativa ssp. indica (XI) and Oryza sativa ssp. japonica (GJ) have recently been updated, providing new opportunities for investigating ongoing gene conversion events and their impact on genome evolution. Results Using comparative genomics and phylogenetic analyses, we evaluated gene conversion rates between duplicated genes produced by polyploidization 100 million years ago (mya) in GJ and XI. At least 5.19–5.77% of genes duplicated across the three rice genomes were affected by whole-gene conversion after the divergence of GJ and XI at ~ 0.4 mya, with more (7.77–9.53%) showing conversion of only portions of genes. Independently converted duplicates surviving in the genomes of different subspecies often use the same donor genes. The ongoing gene conversion frequency was higher near chromosome termini, with a single pair of homoeologous chromosomes, 11 and 12, in each rice genome being most affected. Notably, ongoing gene conversion has maintained similarity between very ancient duplicates, provided opportunities for further gene conversion, and accelerated rice divergence. Chromosome rearrangements after polyploidization are associated with ongoing gene conversion events, and they directly restrict recombination and inhibit duplicated gene conversion between homeologous regions. Furthermore, we found that the converted genes tended to have more similar expression patterns than nonconverted duplicates. Gene conversion affects biological functions associated with multiple genes, such as catalytic activity, implying opportunities for interaction among members of large gene families, such as NBS-LRR disease-resistance genes, contributing to the occurrence of the gene conversion. Conclusion Duplicated genes in rice subspecies generated by grass polyploidization ~ 100 mya remain affected by gene conversion at high frequency, with important implications for the divergence of rice subspecies.

Ewing sarcoma (ES) is a highly malignant primary bone tumor with poor prognosis. Studies have shown that abnormal expression of lncRNA influences the prognosis of tumor patients. Herein, we established that FOXP4-AS1 was up-regulated in ES and this correlated with poor prognosis. Further analysis illustrated that FOXP4-AS1 down-regulation repression growth, migration, along with invasion of ES. On the contrary, up-regulation of FOXP4-AS1 promoted the growth, migration, as well as invasion of ES. To explore the mechanism of FOXP4-AS1, Spearman correlation analysis was carried out to determine genes that were remarkably linked to FOXP4-AS1 expression. The potential functions and pathways involving FOXP4-AS1 were identified by GO analysis, Hallmark gene set enrichment analysis, GSEA, and GSVA. The subcellular fractionation results illustrated that FOXP4-AS1 was primarily located in the cytoplasm of ES cells. Then a ceRNA network of FOXP4-AS1 was constructed. Analysis of the ceRNA network and GSEA yielded two candidate mRNAs for FOXP4-AS1. Results of the combined survival analysis led us to speculate that FOXP4-AS1 may affect the expression of TMPO by sponging miR-298, thereby regulating the malignant phenotype of ES. Finally, we found that FOXP4-AS1 may modulates the tumor immune microenvironment in an extracellular vesicle-mediated manner. In summary, FOXP4-AS1 correlates with poor prognosis of ES. It promotes the growth, migration, as well as invasion of ES cells and may modulate the tumor immune microenvironment.

The thermal shrinkage of commercial polypropylene (PP) separator at high temperature seriously restricts the development of safe and stable lithium-sulfur (Li-S) batteries. To address such issue, herein, we present an interfacial engineering strategy by coating conductive polypyrrole (PPY) modified lithium-montmorillonite (PPY/Li-MMT) on the PP separator. The PPY/Li-MMT can service as an effective heat release layer to enhance the high-temperature stability of Li-S batteries. The engineered PP separator exhibits superior performance: it shows nearly no thermal shrinkage even up to 150 °C. Most importantly, the interface engineered separator has not be punctured during the hot piercing testing and the Li-S batteries deliver outstanding thermal stability even at 80 °C. In addition, the PPY/Li-MMT modified PP separator owns very high electrolyte uptake of 348.6%, large ionic conductivity of 3.63 mS cm−1 and stable performance over 600 cycles. This work provides an effective interfacial engineering strategy by coating PPY/Li-MMT interlayer to significantly improve the electrochemical performance and particularly the high-temperature stability of Li-S batteries.

In order to improve the stability of Cur-LCL and extend the circulation time of the drug in the body, the existing methods of preparing curcumin liposomes (Cur-Lips) and curcumin long-circulating liposomes (Cur-LCL) are optimized. Cur-Lips are prepared by ethanol injection method, and the Cur-LCL is prepared by inserting DSPE-PEG2000 into Cur-Lips by post-insertion. The results show the appearance is a circular vesicular sphere, and the average encapsulation efficiency is (88.91±0.94)%, which stays unchanged at 4 ℃ for 15 days; the average leakage rate is 2.4%, which has good stability; the average particle size is (118.4±3.2) nm (n=3), showing a single peak distribution, and the average potential is (-12.9±0.32) mV (n=3); the dissolution medium is selected according to the solubility standard, and the physiological solution of 1% Tween 80 is selected as the dissolution medium for the in vitro release experiment. Cur APIs are almost completely released at 12 h while Cur-Lips are almost completely released at 36 h, and the cumulative release rate is 92.67%. Cur-LCL is almost completely released at 72 h, and the cumulative release rate is 91.36%. Cur-LCL has obvious function in slowing the release process, which could prolong the circulation time of the drug in the blood and achieve long-circulation effects.

Abstract Inverted planar perovskite solar cells (PSCs), which are regarded as promising devices for new generation of photovoltaic systems, show many advantages, such as low-temperature film formation, low-cost fabrication, and smaller hysteresis compared with those of traditional n-i-p PSCs. As an important carrier transport layer in PSCs, the hole transport layer (HTL) considerably affects the device performance. Therefore, HTL modification becomes one of the most critical issues in improving the performance of PSCs. In this paper, we report an effective and environmentally friendly UV-ozone treatment method to enhance the hydrophilia of reduced graphene oxide (rGO) with its excellent electrical performance. The treated rGO was applied to doped poly(3,4-ethylenedioxythiophene) poly(styrene-sulfonate) (PEDOT:PSS) as HTL material of PSCs. Consequently, the performance of rGO/PEDOT:PSS-doped PSCs was improved significantly, with power conversion efficiency (PCE) of 10.7%, Jsc of 16.75 mA/cm2, Voc of 0.87 V, and FF of 75%. The PCE of this doped PSCs was 27% higher than that of the PSCs with pristine PEDOT:PSS as HTL. This performance was attributed to the excellent surface morphology and optimized hole mobility of the solution-processable rGO-modified PEDOT:PSS.