Temporal shifts in 24 notifiable infectious diseases in China before and during the COVID-19 pandemic.

The coronavirus disease 2019 (COVID-19) pandemic, along with the implementation of public health and social measures (PHSMs), have markedly reshaped infectious disease transmission dynamics. We analysed the impact of PHSMs on 24 notifiable infectious diseases (NIDs) in the Chinese mainland, using time series models to forecast transmission trends without PHSMs or pandemic. Our findings revealed distinct seasonal patterns in NID incidence, with respiratory diseases showing the greatest response to PHSMs, while bloodborne and sexually transmitted diseases responded more moderately. 8 NIDs were identified as susceptible to PHSMs, including hand, foot, and mouth disease, dengue fever, rubella, scarlet fever, pertussis, mumps, malaria, and Japanese encephalitis. The termination of PHSMs did not cause NIDs resurgence immediately, except for pertussis, which experienced its highest peak in December 2023 since January 2008. Our findings highlight the varied impact of PHSMs on different NIDs and the importance of sustainable, long-term strategies, like vaccine development.

Leritrelvir for the treatment of mild or moderate COVID-19 without co-administered ritonavir: a multicentre randomised, double-blind, placebo-controlled phase 3 trial.

Background: Leritrelvir is a novel α-ketoamide based peptidomimetic inhibitor of SARS-CoV-2 main protease. A preclinical study has demonstrated leritrelvir poses similar antiviral activities towards different SARS-CoV-2 variants compared with nirmatrelvir. A phase 2 clinical trial has shown a comparable antiviral efficacy and safety between leritrelvir with and without ritonavir co-administration. This trial aims to test efficacy and safety of leritrelvir monotherapy in adults with mild-to-moderate COVID-19. Methods: This was a randomised, double-blind, placebo-controlled, multicentre phase 3 trial at 29 clinical sites in China. Enrolled patients were from 18 to 75 years old, diagnosed with mild or moderate COVID-19 and not requiring hospitalization. Patients had a positive SARS-CoV-2 nucleic acid test (NAT) and at least one of the COVID-19 symptoms within 48 h before randomization, and the interval between the first positive SARS-CoV-2 NAT and randomization was ≤120 h (5 days). Patients were randomly assigned in a 1:1 ratio to receive a 5-day course of either oral leritrelvir 400 mg TID or placebo. The primary efficacy endpoint was the time from the first dose to sustained clinical recovery of all 11 symptoms (stuffy or runny nose, sore throat, shortness of breath or dyspnea, cough, muscle or body aches, headache, chills, fever ≥37 °C, nausea, vomiting, and diarrhea). The safety endpoint was the incidence of adverse events (AE). Primary and safety analyses were performed in the intention-to-treat (ITT) population. This study is registered with ClinicalTrials.gov, NCT05620160. Findings: Between Nov 12 and Dec 30, 2022 when the zero COVID policy was abolished nationwide, a total of 1359 patients underwent randomization, 680 were assigned to leritrelvir group and 679 to placebo group. The median time to sustained clinical recovery in leritrelvir group was significantly shorter (251.02 h [IQR 188.95-428.68 h]) than that of Placebo (271.33 h [IQR 219.00-529.63 h], P = 0.0022, hazard ratio [HR] 1.20, 95% confidence interval [CI], 1.07-1.35). Further analysis of subgroups for the median time to sustained clinical recovery revealed that (1) subgroup with positive viral nucleic acid tested ≤72 h had a 33.9 h difference in leritrelvir group than that of placebo; (2) the subgroup with baseline viral load >8 log 10 Copies/mL in leritrelvir group had 51.3 h difference than that of placebo. Leritrelvir reduced viral load by 0.82 log10 on day 4 compared to placebo. No participants in either group progressed to severe COVID-19 by day 29. Adverse events were reported in two groups: leritrelvir 315 (46.46%) compared with placebo 292 (43.52%). Treatment-relevant AEs were similar 218 (32.15%) in the leritrelvir group and 186 (27.72%) in placebo. Two cases of COVID-19 pneumonia were reported in placebo group, and one case in leritrelvir group, none of them were considered by the investigators to be leritrelvir related. The most frequently reported AEs (occurring in ≥5% of participants in at least one group) were laboratory finding: hypertriglyceridemia (leritrelvir 79 [11.7%] vs. placebo 70 [10.4%]) and hyperlipidemia (60 [8.8%] vs. 52 [7.7%]); all of them were nonserious. Interpretation: Leritrelvir monotherapy has good efficacy for mild-to-moderate COVID-19 and without serious safety concerns. Funding: This study was funded by the National Multidisciplinary Innovation Team Project of Traditional Chinese Medicine, Guangdong Science and Technology Foundation, Guangzhou Science and Technology Planning Project and R&D Program of Guangzhou Laboratory.

Analysis of the hypovirulent Klebsiella pneumoniae with the NDM-5 gene on IncN plasmids.

IMPORTANCE: It is crucial to strengthen the ongoing clinical surveillance of non-highly virulent, multi-resistant Klebsiella pneumoniae.

Covert dissemination of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae: emergence of low virulence phenotype strains.

This study aimed to explore the epidemic, clinical characteristics, and molecular and virulence attributes of Klebsiella pneumoniae serotype K54 (K54-Kp). A retrospective study was conducted on 328 strains of Klebsiella pneumoniae screened in a Chinese hospital from January 2016 to December 2019. The virulence genes and antibiotic resistance genes (ARGs) were detected by PCR, and a drug sensitivity test was adopted to detect drug resistance. Multilocus sequence typing (MLST) and PFGE were performed to determine the clonal correlation between isolates. Biofilm formation assay, serum complement-mediated killing, and Galleria mellonella infection were used to characterize the virulence potential. Our results showed that thirty strains of K54-Kp were screened from 328 strains of bacteria, with an annual detection rate of 2.29%. K54-Kp had a high resistance rate to antibiotics commonly used in the clinic, and patients with hepatobiliary diseases were prone to K54-Kp infection. MLST typing showed 10 sequence typing, mainly ST29 (11/30), which concentrated in the B2 cluster. K54-Kp primarily carried virulence genes of aerobactin, silS, allS, wcaG, wabG, and mrkD, among which the terW gene was closely related to ST29 (p<0.05). The strains infected by the bloodstream had strong biofilm formation ability (p<0.05). Most strains were sensitive to serum. Still, the virulence of pLVPK-like virulence plasmid in ST29-K54 Klebsiella pneumoniae was lower than that of ST11 type and NTUH-K2044 in the Galleria mellonella model. Therefore, these findings supply a foundation to roundly comprehend K54-Kp, and clinicians should strengthen supervision and attention.

Functional Portrait and Genomic Feature of Carbapenem-Resistant Pseudomonas mendocina Harboring blaNDM-1 and blaIMP-1 in China.

The purpose of this research was to analyze the functional portraits and genomic features of carbapenem-resistant Pseudomonas mendocina carrying NDM-1 and IMP-1. The resistance mechanism of the strain was verified by in vivo experiments. Genomic data were aligned and analyzed in the NCBI database. Growth curve measurements were used to describe the growth characteristics of the bacteria. The virulence of P. mendocina strain was analyzed by serum killing assay and biofilm formation assay. Plasmid conjugation experiments were performed to verify the transferability of plasmids carrying drug-resistance genes. The P. mendocina strain was highly resistant to carbapenems. In addition, ST typing is unknown and has been submitted to Genebank. The strain carried two carbapenemase genes, including NDM-1 and IMP-1. Among them, blaNDM-1 was located on a 5.62832 Mb chromosome, and blaIMP-1 was located on a 172.851 Kb transferable plasmid, which was a very close relative of pIMP-NY7610 in China. The strain also had a variety of virulence genes, which were expressed in the siderophore, capsule, pilus, alginate, flagella, etc. The study suggests that the functional portrait and genomic features of carbapenem-resistant P. mendocina harboring blaNDM-1 and blaIMP-1 are unique to China. This outcome represents antibiotic resistance exhibited in the genus Pseudomonas by acquiring chromosomes and plasmid genes. The monitoring and supervision of antimicrobial usage must be strengthened since the multi-drug-resistant and moderately virulent P. mendocina will attract much attention in the near future.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

Background: The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. Methods: A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Results: Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. Conclusion: In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Four thermostatic steps: A novel CRISPR-Cas12-based system for the rapid at-home detection of respiratory pathogens.

The outbreak of coronavirus disease 2019 (COVID-19) in 2019 has severely damaged the world's economy and public health and made people pay more attention to respiratory infectious diseases. However, traditional quantitative real-time polymerase chain reaction (qRT-PCR) nucleic acid detection kits require RNA extraction, reverse transcription, and amplification, as well as the support of large-scale equipment to enrich and purify nucleic acids and precise temperature control. Therefore, novel, fast, convenient, sensitive and specific detection methods are urgently being developed and moving to proof of concept test. In this study, we developed a new nucleic acid detection system, referred to as 4 Thermostatic steps (4TS), which innovatively allows all the detection processes to be completed in a constant temperature device, which performs extraction, amplification, cutting of targets, and detection within 40 min. The assay can specifically and sensitively detect five respiratory pathogens, namely SARS-CoV-2, Mycoplasma felis (MF), Chlamydia felis (CF), Feline calicivirus (FCV), and Feline herpes virus (FHV). In addition, a cost-effective and practical small-scale reaction device was designed and developed to maintain stable reaction conditions. The results of the detection of the five viruses show that the sensitivity of the system is greater than 94%, and specificity is 100%. The 4TS system does not require complex equipment, which makes it convenient and fast to operate, and allows immediate testing for suspected infectious agents at home or in small clinics. Therefore, the assay system has diagnostic value and significant potential for further reducing the cost of early screening of infectious diseases and expanding its application. KEY POINTS: • The 4TS system enables the accurate and specific detection of nucleic acid of pathogens at 37 °C in four simple steps, and the whole process only takes 40 min. •A simple alkali solution can be used to extract nucleic acid. • A small portable device simple to operate is developed for home diagnosis and detection of respiratory pathogens.

Case report: Severe pneumonia and pneumomediastinum in a previously robust adolescent caused by Omicron BA.5.2.

The manifestation of severe pneumonia is only occasional, and pneumomediastinum is a condition that occurs rarely in Coronavirus disease 2019 (COVID-19) patients, especially in those patients who are infected with the Omicron variant. In addition, whether severe pneumonia or pneumomediastinum often occurs in patients in older age, in poor physical condition, or with underlying diseases remains to be ascertained. To date, severe pneumonia and pneumomediastinum due to Omicron infection had not been reported in a young patient with an excellent physical condition. In this study, we report such a case with the aforementioned manifestations in a robust adolescent infected with Omicron BA.5.2.

Redistribution and Activation of CD16brightCD56dim NK Cell Subset to Fight against Omicron Subvariant BA.2 after COVID-19 Vaccination.

With the alarming surge in COVID-19 cases globally, vaccination must be prioritised to achieve herd immunity. Immune dysfunction is detected in the majority of patients with COVID-19; however, it remains unclear whether the immune responses elicited by COVID-19 vaccination function against the Omicron subvariant BA.2. Of the 508 enrolled patients infected with Omicron BA.2, 102 were unvaccinated controls, and 406 were vaccinated. Despite the presence of clinical symptoms in both groups, vaccination led to a significant decline in nausea or vomiting, abdominal pain, headache, pulmonary infection, and overall clinical symptoms and a moderate rise in body temperature. The individuals infected with Omicron BA.2 were also characterised by a mild increase in both serum pro- and anti-inflammatory cytokine levels after vaccination. There were no significant differences or trend changes between T- and B-lymphocyte subsets; however, a significant expansion of NK lymphocytes in COVID-19-vaccinated patients was observed. Moreover, the most effective CD16brightCD56dim subsets of NK cells showed increased functional capacities, as evidenced by a significantly greater IFN-γ secretion and a stronger cytotoxic potential in the patients infected with Omicron BA.2 after vaccination. Collectively, these results suggest that COVID-19 vaccination interventions promote the redistribution and activation of CD16brightCD56dim NK cell subsets against viral infections and that they could facilitate the clinical management of patients infected with Omicron BA.2.

Important oncogenic and immunogenic roles of SPP1 and CSF1 in hepatocellular carcinoma.

The treatment and prognosis of liver cancer remain the focus of medical research. Studies have shown that SPP1 and CSF1 play important roles in cell proliferation, invasion, and metastasis. Therefore, this study analyzed the oncogenic and immunologic roles of SPP1 and CSF1 in hepatocellular carcinoma (HCC). We found that the expression levels of SPP1 and CSF1 in HCC were markedly increased and positively correlated. High SPP1 expression was significantly associated with poor OS, DSS, PFS, and RFS. It was not affected by gender, alcohol use, HBV, or race, whereas CSF1 was affected by these factors. Higher expression levels of SPP1 and CSF1 indicated higher levels of immune cell infiltration and a higher immune score with the R software package ESTIMATE. Further analysis revealed that many genes work co-expressed between SPP1 and CSF1 with the LinkedOmics database, which were mainly involved in signal transduction, the integral components of the membrane, protein binding, and osteoclast differentiation. In addition, we screened ten hub genes using cytoHubba, among which the expression of four genes was significantly associated with the prognosis of HCC patients. Finally, we demonstrated the oncogenic and immunologic roles of SPP1 and CSF1 using the vitro experiments. Reducing the expression of either SPP1 or CSF1 could significantly reduce the proliferation of HCC cells and the expression of CSF1, SPP1, and the other four hub genes. This study suggested that SPP1 and CSF1 interact with each other and have the potential to be therapeutic and prognostic targets for HCC.

Horizontal gene transfer via OMVs co-carrying virulence and antimicrobial-resistant genes is a novel way for the dissemination of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Introduction: The rapidly increased isolation rate of CR-HvKP worldwide has brought great difficulties in controlling clinical infection. Moreover, it has been demonstrated that the transmission of drug-resistant genes among bacteria can be mediated by outer membrane vesicles (OMVs), which is a new way of horizontal gene transfer (HGT). The transmission of virulence genes among bacteria has also been well studied; however, it remains unclear whether virulence and drug-resistant genes can be co-transmitted simultaneously. Co-transmission of virulence and drug-resistant genes is essential for the formation and prevalence of CR-HvKP. Methods: First, we isolated OMVs from CR-HvKP by cushioned-density gradient ultracentrifugation (C-DGUC). TEM and DLS were used to examine the morphology and size of bacterial OMVs. OMV-mediated gene transfer in liquid cultures and the acquisition of the carbapenem gene and virulence gene was confirmed using colony-PCR. Antimicrobial susceptibility testing, mCIM and eCIM were conducted for the resistance of transformant. Serum killing assay, assessment of the anti-biofilm effect and galleria mellonella infection model, mucoviscosity assay, extraction and quantification of capsules were verified the virulence of transformant. Pulsed-field gel electrophoresis (PFGE), S1 nuclease-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting hybridization confirmed the plasmid of transformant. Results: Firstly, OMVs were isolated from CR-HvKP NUHL30457 (K2, ST86). TEM and DLS analyses revealed the spherical morphology of the vesicles. Secondly, our study demonstrated that CR-HvKP delivered genetic material, incorporated DNA within the OMVs, and protected it from degradation by extracellular exonucleases. Thirdly, the vesicular lumen DNA was delivered to the recipient cells after determining the presence of virulence and carbapenem-resistant genes in the CR-HvKP OMVs. Importantly, S1-PFGE and Southern hybridization analysis of the 700603 transformant strain showed that the transformant contained both drug-resistant and virulence plasmids. Discussion: In the present study, we aimed to clarify the role of CRHvKP-OMVs in transmitting CR-HvKP among K. pneumoniae. Collectively, our findings provided valuable insights into the evolution of CR-HvKP.

CRISPR-Cas13: A new technology for the rapid detection of pathogenic microorganisms.

Pathogenic microorganisms have major impacts on human lives. Rapid and sensitive diagnostic tools are urgently needed to facilitate the early treatment of microbial infections and the effective control of microbial transmission. CRISPR-Cas13 employs programmable RNA to produce a sensitive and specific method with high base resolution and thus to provide a novel tool for the rapid detection of microorganisms. The review aims to provide insights to spur further development by summarizing the characteristics of effectors of the CRISPR-Cas13 system and by describing the latest research into its application in the rapid detection of pathogenic microorganisms in combination with nucleic acid extraction, isothermal amplification, and product detection.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10-5 and 8.7× 10-7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

Construction and validation of a prognostic risk model for breast cancer based on protein expression.

Breast cancer (BRCA) is the primary cause of mortality among females globally. The combination of advanced genomic analysis with proteomics characterization to construct a protein prognostic model will help to screen effective biomarkers and find new therapeutic directions. This study obtained proteomics data from The Cancer Proteome Atlas (TCPA) dataset and clinical data from The Cancer Genome Atlas (TCGA) dataset. Kaplan-Meier and Cox regression analyses were used to construct a prognostic risk model, which was consisted of 6 proteins (CASPASE7CLEAVEDD198, NFKBP65-pS536, PCADHERIN, P27, X4EBP1-pT70, and EIF4G). Based on risk curves, survival curves, receiver operating characteristic curves, and independent prognostic analysis, the protein prognostic model could be viewed as an independent factor to accurately predict the survival time of BRCA patients. We further validated that this prognostic model had good predictive performance in the GSE88770 dataset. The expression of 6 proteins was significantly associated with the overall survival of BRCA patients. The 6 proteins and encoding genes were differentially expressed in normal and primary tumor tissues and in different BRCA stages. In addition, we verified the expression of 3 differential proteins by immunohistochemistry and found that CDH3 and EIF4G1 were significantly higher in breast cancer tissues. Functional enrichment analysis indicated that the 6 genes were mainly related to the HIF-1 signaling pathway and the PI3K-AKT signaling pathway. This study suggested that the prognosis-related proteins might serve as new biomarkers for BRCA diagnosis, and that the risk model could be used to predict the prognosis of BRCA patients.

miR-146b-5p activation of hepatic stellate cells contributes to the progression of fibrosis by directly targeting HIPK1.

The present study aimed to explore the biological functions of microRNA (miR)-146b-5p and homeodomain interacting protein kinase 1 (HIPK1) in the progression of hepatic fibrosis (HF) and to identify the underlying mechanism. A rat HF model was established by administering a subcutaneous injection of carbon tetrachloride (CCl4). Relative levels of miR-146b-5p and HIPK1 in fibrotic rat liver tissues and the rat hepatic stellate cell (HSC) line HSC-T6 were measured by quantitative reverse transcription PCR, western blotting and immunohistochemistry. Following activation of HSC-T6 cells by lipopolysaccharide (LPS) induction, cell viability was examined by MTT assay. Transfection of miR-146b-5p mimic or inhibitor into HSC-T6 cells was performed, with the aim to identify the influence of miR-146b-5p on HSC-T6 cell behavior. The targeting relationship between miR-146b-5p and HIPK1 was predicted by TargetScan 7.2 and StarBase 3.0 and it was later verified by a dual-luciferase reporter assay. Through lentivirus transfection, the biological function of HIPK1 in regulating the progression of HF and the underlying mechanism were investigated. The results showed that miR-146b-5p was upregulated in liver tissues of rats with HF and activated HSC-T6 cells, while HIPK1 was downregulated in liver tissues of rats with HF and activated HSC-T6 cells. miR-146b-5p was able to upregulate the activation markers of LPS-induced HSC-T6 cells, upregulate COL1A1 and TGF-ß, increase cell viability and contribute to fibrosis progression. HIPK1 was validated as the direct target of miR-146b-5p and its overexpression could effectively reduce the effect of miR-146b-5p in contribution to the progression of HF. In conclusion, miR-146b-5p was significantly upregulated during the progression of HF. By targeting and downregulating HIPK1, miR-146b-5p could significantly activate HSCs, upregulate COL1A1 and TGF-ß and contribute to fibrosis progression. miR-146b-5p is a potential biomarker and therapeutic target for HF.

Nosocomial transmission and rearrangement of large resistance-virulence hybrid plasmids between two bacteremic ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains with low fitness cost.

OBJECTIVES: To characterize nosocomial transmission and rearrangement of the resistance-virulence plasmid between two ST11-K64 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains (JX-CR-hvKP-10 and JX-CR-hvKP-9) with low fitness. METHODS: Phenotypic tests were used to assess the virulence of JX-CR-hvKP-10 and JX-CR-hvKP-9. Whole-genome sequencing was used to analyze JX-CR-hvKP-10 and JX-CR-hvKP-9 chromosomes and plasmids. Fitness and conjugation experiments were also conducted using these two CR-hvKP isolates. RESULTS: Phenotypic tests indicated that both JX-CR-hvKP-10 and JX-CR-hvKP-9 were multidrug-resistant and hypervirulent K. pneumoniae. Whole-genome sequencing and clinical information demonstrated that the super large resistance-virulence fusion plasmid pJX10-1 formed precisely by the fusion of pJX9-1 and pJX9-2 via the nosocomial transmission. Interestingly pJX9-1 itself was also a classic resistance-virulence fusion plasmid by way of the blaKPC-carrying resistance plasmid and pLVPK-like virulence plasmid. Compared with classic K. pneumoniae ATCC700603, fitness analysis revealed no significant difference in growth was observed between JX-CR-hvKP-10 and JX-CR-hvKP-9. CONCLUSION: Nosocomial transmission and rearrangement of a blaKPC-harboring plasmid and a pLVPK-like virulence plasmid with a low fitness cost in ST11 K. pneumoniae enhances drug resistance and virulence simultaneously. Thus, active surveillance of this hybrid plasmid is needed to prevent these efficient resistance-virulence plasmids from disseminating in hospital settings.

Secreted phosphoprotein 1 as a potential prognostic and immunotherapy biomarker in multiple human cancers.

Secreted phosphoprotein 1 (SPP1) is involved in immune regulation, cell survival, and tumor progression. Studies have demonstrated that SPP1 plays an important role in certain individual tumors. However, the expression profile and oncogenic features of SPP1 in diverse cancers are remaining unknown. Therefore, we performed a comprehensive analysis using The Cancer Genome Atlas (TCGA) database. Raw data of 33 cancer types were download from the University of California Santa Cruz (UCSC) Xena website. The expression of SPP1 and its relationship with tumor prognosis, immune invasion, tumor microenvironment, and immunotherapy were analyzed using the R language. The function analysis was conducted using Gene Set Enrichment Analysis (GSEA). The oncogenic features of SPP1 was validated by wound-healing assay and EdU staining assay. SPP1 highly expressed in most cancers. The expression of SPP1 was significant related to prognosis, tumor mutation burden (TMB), microsatellite instability (MSI), and immune checkpoint genes, suggested that SPP1 plays an essential role in the tumor immune microenvironment and immune cell infiltration. The immune/stromal scores correlated positively with the SPP1 expression, and the relationship was affected by tumor heterogeneity and immunotherapy. In addition, SPP1 could predict the response of tumor immunotherapy. Functional analysis revealed the SPP1-associated terms and pathways. Finally, SPP1 significantly elevated cell proliferation and migration in A549, Huh7, HT-29, A2780 tumor cell lines. In conclusion, this study indicated that SPP1 involved in tumorigenesis, tumor progression, and regulated tumor immune microenvironment, revealing SPP1 might be a potential target for evaluating prognosis and immunotherapy in multiple cancers.

Distribution of type VI secretion system (T6SS) in clinical Klebsiella pneumoniae strains from a Chinese hospital and its potential relationship with virulence and drug resistance.

OBJECTIVES: The type VI secretion system (T6SS) in Klebsiella pneumoniae strains isolated from the bloodstream, intestinal, the pyogenic liver abscess has been reported. Here we aimed to characterize T6SS in 248 Klebsiella pneumoniae isolates with all kinds of specimens from a Chinese hospital and to investigate the potential association of T6SS with virulence and drug resistance. METHODS: T6SS genes, capsular serotyping genes, drug resistance genes, and virulence genes were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities were examined by the disk diffusion method. To assess biofilm formation of these clinical Klebsiella pneumoniae isolates, 96-well microtiter plate assays were performed. MLST was used to analyze the genotypes of these Klebsiella pneumoniae isolates. RESULTS: The frequency of T6SS genes among the clinical Klebsiella pneumoniae isolates was 72.2%. The T6SS-positive isolates displayed higher resistance to piperacillin-tazobactam, ciprofloxacin, levofloxacin, meropenem than the T6SS-negative isolates (P < 0.05). The T6SS-positive isolates formed significantly more biofilm mass than the T6SS-negative isolates (mean ± standard deviation [SD], 0.3 ± 0.09 vs.0.16 ± 0.06; P < 0.01). Compared to the T6SS-negative isolates, the T6SS-positive isolates had a higher frequency of virulence genes (rmpA, fimH, entB, kfu, ybtS) and the pLVPK-like plasmid (P < 0.05). CONCLUSION: In conclusion, the prevalence of the type VI secretion system is high in clinical Klebsiella pneumoniae isolates in a Chinese teaching hospital. T6SS-positive strains show higher biofilm-forming activity with high drug resistance and exhibit higher virulence potential.

Prevalence of the NTEKPC-I on IncF Plasmids Among Hypervirulent Klebsiella pneumoniae Isolates in Jiangxi Province, South China.

Infection caused by carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has become a tricky health care threat in China and KPC-2 enzyme is a main factor mediating resistance to carbapenems of K. pneumoniae. Here, we report the characterization of the genetic environment of the blaKPC-2 gene in CR-hvKP clinical isolates from South China. Forty-five non-duplicated CR-hvKP isolates collected in Jiangxi Province from 2018 to 2019 were analyzed. Each of them were multidrug-resistant due to the presence not only of blaKPC-2 gene but also of other resistance determinants, including Metallo-ß-lactamases (NDM-1), extended-spectrum ß-lactamases (TEM-1, CTX-M-14, SHV-1), and plasmid-mediated quinolone resistance determinants (qnrS, aac(6')-Ib-cr). After plasmid analyses of PCR-based replicon typing (PBRT), mapping PCR, amplicon sequencing, and whole-genome sequencing (WGS) were used to analyze the genetic environment of the blaKPC-2 gene. PCR analysis of pLVPK-like plasmids, Southern Blot, and mouse lethality assay were used to characterize the virulence phenotype of K. pneumoniae. Multilocus sequence typing (MLST) analysis showed ST11 CR-hvKP was the predominant clone. In conclusion, this is the first analysis of diverse genetic structures blaKPC-2 gene in CR-hvKP isolates from south China. Both the NTEKPC-I on the IncF plasmids and pLVPK-like virulence plasmids make contributions to the formation of CR-hvKP especially ST11 which need more attention.