Cefoperazone/sulbactam has been shown to be efficacious for the treatment of infections caused by Acinetobacter baumannii; however, the mechanism underlying resistance to this synergistic combination is not well understood. In the present study, two A. baumannii isolates, AB1845 and AB2092, were isolated from a patient with hospital-acquired pneumonia before and after 20 days of cefoperazone/sulbactam therapy (2:1, 3 g every 8 h with a 1-h infusion). The minimum inhibitory concentration (MIC) of cefoperazone/sulbactam for AB1845 and AB2092 was 16/8 and 128/64 mg/L, respectively. Blood samples were collected on day 4 of the treatment to determine the concentration of cefoperazone and sulbactam. The pharmacokinetic/pharmacodynamic (PK/PD) indices (%T>MIC) were calculated to evaluate the dosage regimen and resistance development. The results showed that %T>MIC of cefoperazone and sulbactam was 100% and 34.5% for AB1845, and 0% and 0% for AB2092, respectively. Although there was no available PK/PD target for sulbactam, it was proposed that sulbactam should be administered at higher doses or for prolonged infusion times to achieve better efficacy. To investigate the mechanism of A. baumannii resistance to the cefoperazone/sulbactam combination in vivo, whole-genome sequencing of these two isolates was further performed. The sequencing results showed that 97.6% of the genome sequences were identical and 33 non-synonymous mutations were detected between AB1845 and AB2092. The only difference of these two isolates was showed in sequencing coverage comparison. There was a 6-kb amplified DNA fragment which was three times higher in AB2092, compared with AB1845. The amplified DNA fragment containing the bla OXA-23 gene on transposon Tn2009. Further quantitative real-time PCR results demonstrated that gene expression at the mRNA level of bla OXA-23 was >5 times higher in AB2092 than in AB1845. These results suggested that the bla OXA-23 gene had higher expression level in AB2092 via gene amplification and following transcription. Because gene amplification plays a critical role in antibiotic resistance in many bacteria, it is very likely that the bla OXA-23 amplification results in the development of cefoperazone/sulbactam resistance in vivo.

The objective of this study was to assess the impact of diverse plasmids bearing colistin resistance gene mcr-1 on host fitness. Forty-seven commensal E. coli isolates recovered from the pig farm where mcr-1 was first identified were screened for mcr-1. mcr-1-bearing plasmids were characterized by sequencing. The fitness impact of mcr-1-bearing plasmids was evaluated by in vitro competition assays. Twenty-seven (57.5%) E. coli isolates were positive for mcr-1. The mcr-1 genes were mainly located on plasmids belonging to IncI2 (n = 5), IncX4 (n = 11), IncHI2/ST3 (n = 8), IncFII (n = 2), and IncY (n = 2). InHI2 plasmids also carried other resistance genes (floR, blaCTX-M, and fosA3) and were only detected in isolates from nursery pigs. Sequences of the representative mcr-1-bearing plasmids were almost identical to those of the corresponding plasmid types reported previously. An increase in the fitness of IncI2- and IncX4-carrying strains was observed, while the presence of IncHI2, IncFII and IncY plasmids showed a fitness cost although an insignificant fitness increase was initially observed in IncFII or IncY plasmids-containing strains. Acquisition of IncI2-type plasmid was more beneficial for host E. coli DH5α than either IncHI2 or IncX4 plasmid, while transformants with IncHI2-type plasmid presented a competitive disadvantage against IncI2 or IncX4 plasmid containing strains. In conclusion, IncI2, IncX4, and IncHI2 were the major plasmid types driving the dissemination of mcr-1 in this farm. Increased fitness or co-selection by other antimicrobials might contribute to the further dissemination of the three epidemic mcr-1-positive plasmids (IncI2, IncX4, and IncHI2) in this farm and worldwide.

Bio-production of cadaverine from cheap carbon sources for synthesizing bio-based polyamides is becoming more common. Here, a novel fermentation process for cadaverine bio-production from glucose was implemented by using a microbial consortium of two engineered Escherichia coli strains to relieve the toxic effect of cadaverine on fermentation efficiency. To achieve controllable growth of strains in the microbial consortium, two engineered E. coli strains grown separately on different carbon sources were first constructed. The strains were, an L-lysine-producing E. coli NT1004 with glucose as carbon source, and a cadaverine-producing E. coli CAD03 with glucose metabolism deficiency generated by modifying the PTSGlc system with CRISPR-Cas9 technology and inactivating cadaverine degradation pathways. Co-culturing these two engineered E. coli strains with a mixture of glucose and glycerol led to successful production of cadaverine. After optimizing cultivation conditions, a cadaverine titer of 28.5 g/L was achieved with a multi-stage constant-speed feeding strategy.

Continuous rain and an abnormally wet climate during harvest can easily lead to soybean plants being damaged by field mold (FM), which can reduce seed yield and quality. However, to date, the underlying pathogen and its resistance mechanism have remained unclear. The objective of the present study was to investigate the fungal diversity of various soybean varieties and to identify and confirm the FM pathogenic fungi. A total of 62,382 fungal ITS1 sequences clustered into 164 operational taxonomic units (OTUs) with 97% sequence similarity; 69 taxa were recovered from the samples by internal transcribed spacer (ITS) region sequencing. The fungal community compositions differed among the tested soybeans, with 42 OTUs being amplified from all varieties. The quadratic relationships between fungal diversity and organ-specific mildew indexes were analyzed, confirming that mildew on soybean pods can mitigate FM damage to the seeds. In addition, four potentially pathogenic fungi were isolated from FM-damaged soybean fruits; morphological and molecular identification confirmed these fungi as Aspergillus flavus, A. niger, Fusarium moniliforme, and Penicillium chrysogenum. Further re-inoculation experiments demonstrated that F. moniliforme is dominant among these FM pathogenic fungi. These results lay the foundation for future studies on mitigating or preventing FM damage to soybean.

Osteopontin (OPN) is a multifunctional matricellular protein produced by a broad range of cells including osteoclasts, macrophages, T cells, endothelial cells, and vascular smooth muscle cells. OPN modulates various physiological and pathological events such as inflammation, wound healing, and bone formation and remodeling. Dengue virus (DENV) infection causes an increase in plasma OPN levels, which is correlated with the severity of symptoms and coagulation abnormalities. DENV infection also induces OPN gene expression in human macrophages. This study investigated the inhibitory effects of brefelamide and its methyl ether derivative on DENV-3 by measuring changes in OPN levels in human THP-1 and 293T cell lines infected at different multiplicities of infection and post-infection time points. OPN mRNA expression and viral RNA were detected by reverse transcriptase quantitative real-time PCR, whereas protein level was determined by enzyme-linked immunosorbent assay. We found that viral copy number was higher in 293T than in THP-1 cells. However, THP-1 constitutively expressed higher levels of OPN mRNA and protein, which were enhanced by DENV-3 infection. Brefelamide and its derivative suppressed OPN production in DENV-3 infected THP-1 cells; the effective doses of these compounds had no effect on uninfected cells, indicating low cytotoxicity. These results suggest that brefelamide and its methyl ether derivative have therapeutic effects in preventing inflammation, coagulopathy, and fibrinolysis caused by OPN upregulation induced by DENV-3 infection.

Influenza A virus (IAV) has developed elegant strategies to utilize cellular proteins and pathways to promote replication and evade the host antiviral response. Identification of these sabotaged host factors could increase the number of potential antiviral drug targets. Here, IAV A/PR/8/34 (PR8)- and A/California/04/2009-infected A549 and 293T cells displayed differential virus replication. To determine the host cellular responses of A549 and 293T cells to IAV infection, RNA-seq was used to identify differentially expressed genes. Our data revealed that IAV-infected A549 cells activated stronger virus-sensing signals and highly expressed cytokines, which play significant roles in initiating the innate immune and inflammatory responses. In addition, IAV-infected 293T cells displayed weak immune signaling and cytokine production. Remarkably, IL-17A and associated genes were highly enriched in IAV-infected 293T cells. Furthermore, IL-17A can partially facilitate A549 cell infection by the PR8 strain and PR8-infected IL-17A knock-out mice consistently exhibited decreased weight loss and reduced lung immunopathology, as compared to controls. This work uncovered the differential responses of cells infected with two H1N1 IAV strains and the potential roles of IL-17A in modulating virus infection.

Phosphorylation and dephosphorylation of nucleoprotein (NP) play significant roles in the life cycle of influenza A virus (IAV), and the biological functions of each phosphorylation site on NP are not exactly the same in controlling viral replication. Here, we identified tyrosine 78 residue (Y78) of NP as a novel phosphorylation site by mass spectrometry. Y78 is highly conserved, and the constant NP phosphorylation mimicked by Y78E delayed NP nuclear export through reducing the binding of NP to the cellular export receptor CRM1, and impaired virus growth. Furthermore, the tyrosine kinase inhibitors Dasatinib and AG490 reduced Y78 phosphorylation and accelerated NP nuclear export, suggesting that the Janus and Src kinases-catalyzed Y78 phosphorylation regulated NP nuclear export during viral replication. More importantly, we found that the NP phosphorylation could suppress NP ubiquitination via weakening the interaction between NP and E3 ubiquitin ligase TRIM22, which demonstrated a cross-talk between the phosphorylation and ubiquitination of NP. This study suggests that the phosphorylation status of Y78 regulates IAV replication by inhibiting the nuclear export and ubiquitination of NP. Overall, these findings shed new light on the biological roles of NP phosphorylation, especially its negative role in NP ubiquitination.

Accumulating evidence has indicated that the multiple drug resistant Vibrio parahaemolyticus may pose a serious threat to public health and economic concerns for humans globally. Here, two lytic bacteriophages, namely vB_VpS_BA3 and vB_VpS_CA8, were isolated from sewage collected in Guangzhou, China. Electron microscopy studies revealed both virions taxonomically belonged to the Siphoviridae family with icosahedral head and a long non-contractile tail. The double-stranded DNA genome of phage BA3 was composed of 58648 bp with a GC content of 46.30% while phage CA8 was 58480 bp with an average GC content of 46.42%. In total, 85 putative open reading frames (ORFs) were predicted in the phage BA3 genome while 84 were predicted in that of CA8. The ORFs were associated with phage structure, packing, host lysis, DNA metabolism, and additional functions. Furthermore, average nucleotide identity analysis, comparative genomic features and phylogenetic analysis revealed that BA3 and CA8 represented different isolates but novel members of the family, Siphoviridae. Regarding the host range of the 61 V. parahaemolyticus isolates, BA3 and CA8 had an infectivity of 8.2 and 36.1%, respectively. Furthermore, ∼100 plaque-forming units (pfu)/cell for phage BA3 and ∼180 pfu/cell for phage CA8 were determined to be the viral load under laboratory growth conditions. Accordingly, the phage-killing assay in vitro revealed that phage CA8 achieved approximately 3.65 log unit reductions. The present results indicate that CA8 is potentially applicable for biological control of multidrug resistant V. parahaemolyticus.

Listeria monocytogenes (Lm) is a foodborne zoonotic pathogen that causes listeriosis with a mortality rate of 20-30%. Serovar 4b and 1/2b isolates account for most of listeriosis outbreaks, however, no listeriosis vaccine is available for either prophylactic or therapeutic use. Here, we developed a triple-virulence-genes deletion vaccine strain, and evaluated its safety, immunogenicity, and cross-protective efficiency. The virulence of NTSNΔactA/plcB/orfX was reduced 794-folds compared with the parental strain. Additionally, it was completely eliminated in mice at day 7 post infection and no obvious pathological changes were observed in the organs of mice after prime-boost immunization for 23 days. These results proved that the safety of the Lm vaccine strain remarkably increased. More importantly, the NTSNΔactA/plcB/orfX strain stimulated higher anti-Listeriolysin O (LLO) antibodies, induced significantly higher expression of IFN-γ, TNF-α, IL-17, and IL-6 than the control group, and afforded 100% protection against serovar 4b and 1/2b challenges. Taken together, our research demonstrates that the triple-genes-deletion vaccine has high safety, can elicit strong Th1 type immune response, and affords efficient cross-protection against two serovar Lm strains. It is a promising vaccine for prevention of listeriosis.

SARS-CoV-2 has caused a global health disaster with millions of death worldwide, and the substantial proportion of asymptomatic carriers poses a huge threat to public health. The long-term antibody responses and neutralization activity during natural asymptomatic SARS-CoV-2 infection are unknown. In this study, we used enzyme-linked immunosorbent assays (ELISA) and neutralization assay with purified SARS-CoV-2S and N proteins to study the antibody responses of 156 individuals with natural asymptomatic infection. We found robust antibody responses to SARS-CoV-2 in 156 patients from 6 to 12 months. Although the antibody responses gradually decreased, S-IgG was more stable than N-IgG. S-IgG was still detected in 79% of naturally infected individuals after 12 months of infection. Moderate to potent neutralization activities were also observed in 98.74% of patients 6 months after infection. However, this proportion decreased at 8-month (46.15%) and 10-month (39.11%) after infection, respectively. Only 23.72% of patients displayed potent neutralization activity at 12 months. This study strongly supports the long-term presence of antibodies against SARS-CoV-2 in individuals with natural asymptomatic infection, although the magnitude of the antibody responses started to cripple 6 months after infection.

This study aimed to investigate the prevalence of fosfomycin fosA7 in Salmonella enterica isolates from food animals and retail meat products in China and the impact of fosA7 on bacterial fitness. A total of 360 Salmonella isolates collected from 11 provinces and cities in China were detected for fosA7. All fosA7-positive Salmonella isolates were determined minimum inhibitory concentrations (MICs) and sequenced by Illumina Hiseq. The fosA7 gene of S. Derby isolate HA2-WA5 was knocked out. The full length of fosA7 was cloned into vector pBR322 and then transformed into various hosts. MICs of fosfomycin, growth curves, stability, and fitness of fosA7 were evaluated. The fosA7 gene was identified in S. Derby (ST40, n = 30) and S. Reading (ST1628, n = 5). MICs to fosfomycin of 35 fosA7-positive isolates were 1 to 32 mg/L. All fosA7 were located on chromosomes of Salmonella. The deletion of fosA7 in HA2-WA5 decreased fosfomycin MIC by 16-fold and slightly affected its fitness. The acquisition of plasmid-borne fosA7 enhanced MICs of fosfomycin in Salmonella (1,024-fold) and Escherichia coli (16-fold). The recombinant plasmid pBR322-fosA7 was stable in Salmonella Typhimurium, S. Pullorum, S. Derby, and E. coli, except for Salmonella Enteritidis, and barely affected on the growth of them but significantly increased biological fitness in Salmonella. The spread of specific Salmonella serovars such as S. Derby ST40 will facilitate the dissemination of fosA7. fosA7 can confer high-level fosfomycin resistance and enhance bacterial fitness in Salmonella if transferred on plasmids; thus, it has the potential to be a reservoir of the mobilized fosfomycin resistance gene.

Deinococcus radiodurans is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported that the pattern of alteration in Deinococcus proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of Staphylococcus aureus was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing S. aureus colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from D. radiodurans and investigated their inhibitory effects on S. aureus colonization and biofilm formation in vitro and in vivo. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δdra0033 strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with S. aureus biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-N-acetylglucosamine (PNAG), a key molecule required for S. aureus biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of S. aureus biofilm formation by D. radiodurans. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with S. aureus biofilms.

Long-interspersed element 1 (LINE-1) is an autonomous non-LTR retrotransposon. Its replication can cause mutation and rearrangement of host genomic DNA, which may result in serious genetic diseases. Host cells therefore developed defense strategies to restrict LINE-1 mobilization. In this study, we reported that CCHC-type zinc-finger protein ZCCHC3 can repress LINE-1 retrotransposition, and this activity is closely related to its zinc-finger domain. Further studies show that ZCCHC3 can post-transcriptionally diminish the LINE-1 RNA level. The association of ZCCHC3 with both LINE-1 RNA and ORF1 suggests that ZCCHC3 interacts with LINE-1 RNP and consequently causes its RNA degradation. These data demonstrate collectively that ZCCHC3 contributes to the cellular control of LINE-1 replication.

MicroRNAs (miRNAs) play a vital role in tuberculosis (TB). Vitamin D receptor (VDR), an miRNA target gene, and its ligand, vitamin D3 (VitD3), have been reported to exert protective effects against TB. However, whether miRNAs can affect the progression of TB by targeting VDR has not been reported.

Pseudomonas aeruginosa is the most relevant pathogen to the severe exacerbations of patients with chronic obstructive pulmonary disease (COPD). However, the genetic and functional characteristics of P. aeruginosa isolates from COPD airways still remain less understood. In this study, the genetic, phylogenetic, phenotypic, and transcriptional features of P. aeruginosa isolates from COPD sputa were comprehensively explored by susceptibility testing, comparative-genomic analysis, phylogenetic analysis, phenotypic profiling, and comparative-transcriptomic analysis. We found that P. aeruginosa was prevalent in elder COPD patients and highly resisted to many commonly used antibiotics. P. aeruginosa COPD isolates harbored a substantial number of variant sites that might influence the primary metabolism and substance transport system. These isolates were discretely distributed in the phylogenetic tree and clustered with internationally collected P. aeruginosa in two major groups, and could be classified into three groups according to their differences in virulence-related phenotypes. Furthermore, the transcriptional patterns of COPD isolates could be classified into PAO1-like group with reduced protein secretion and motility and PAO1-distinct group with decreased substance transport but enhanced primary metabolism. In conclusion, this study demonstrates that P. aeruginosa isolates from COPD patients have abundant genetic and phenotypic diversity, and provides an important reference for further exploring the survival strategy of P. aeruginosa in COPD airways and the development of anti-pseudomonal therapy.

The deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known regarding the mechanisms of viral infection and replication due to the lack of immortalized honey bee cell lines. To solve this problem, we established an in vitro system using honey bee pupal tissue to reconstruct DWV binding and entry into the host cell, followed by translation of the RNA genome and polyprotein processing using RNA-dependent RNA polymerase (RdRP) as a marker. Using this system, the P-domain of the virion subunit VP1 was found to be essential for DWV infection, but not for binding and entry into the cell. DWV efficiently infected the head tissue derived from early but not late pupa, suggesting that undifferentiated cells are targeted for viral infection. Furthermore, we found that inhibitors of mammalian picornavirus 3C-protease, rupintrivir and quercetin suppressed RdRP synthesis, indicating that this in vitro system is also useful for screening a compound to control viral infection. Our in vitro system may help to understand the mechanism of DWV infection in host cells.

Vibrio alginolyticus is one of the most important of pathogens that can infect humans and a variety of aquatic animals, and it can cause food poisoning and septicemia in humans. Widely used antibiotics are gradually losing their usefulness, and phages are gaining more attention as new antibacterial strategies. To have more potential strategies for controlling pathogenic bacteria, we isolated a novel V. alginolyticus phage BUCT549 from seafood market sewage. It was classified as a new member of the family Siphoviridae by transmission electron microscopy and a phylogenetic tree. We propose creating a new genus for BUCT549 based on the intergenomic similarities (maximum is 56%) obtained from VIRIDIC calculations. Phage BUCT549 could be used for phage therapy due to its stability in a wide pH (3.0-11.0) range and high-temperature (up to 60°C) environment. It had a latent period of 30-40 min and a burst size of 141 PFU/infected bacterium. In the phylogenetic tree based on a terminase large subunit, BUCT549 was closely related to eight Vibrio phages with different species of host. Meanwhile, our experiments proved that BUCT549 has the ability to infect a strain of Vibrio parahaemolyticus. A coevolution experiment determined that three strains of tolerant V. alginolyticus evaded phage infestation by mutating the MSHA-related membrane protein expression genes, which caused the loss of flagellum. This research on novel phage identification and the mechanism of infestation will help phages to become an integral part of the strategy for biological control agents.

Influenza virus RNA-dependent RNA polymerase (vRdRp) does not have capping activity and relies on the capped RNAs produced by the host RNA polymerase II (RNAPII). The viral polymerases process the capped RNAs to produce short capped RNA fragments that are used as primers to initiate the transcription of viral mRNAs. This process, known as cap-snatching, can be targeted by antiviral therapeutics. Here, anthralin was identified as an inhibitor against influenza a virus (IAV) infection by targeting the cap-snatching activity of the viral polymerase. Anthralin, an FDA-approved drug used in the treatment of psoriasis, shows antiviral activity against IAV infection in vitro and in vivo. Importantly, anthralin significantly reduces weight loss, lung injury, and mortality caused by IAV infection in mice. The mechanism of action study revealed that anthralin inhibits the cap-binding function of PB2 subunit and endonuclease activity of PA. As a result, viral mRNA transcription is blocked, leading to the decreases in viral RNA replication and viral protein expression. In conclusion, anthralin has been demonstrated to have the potential of an alternative antiviral against influenza virus infection. Also, targeting the captive pocket structure that includes the N-terminus of PA endonuclease domain and the C-terminal of PB2 cap-binding domain of IAV RdRp may be an excellent strategy for developing anti-influenza drugs.

In this study, attention has been focused on the ecology of yeasts during the spontaneous and inoculated fermentation processes of Vidal blanc icewine in northeast China, which is very important for screening autochthonous yeast strains, understanding the roles of these strains, and managing fermentation. The strategies were to conduct spontaneous and inoculated laboratory-scale fermentation processes simultaneously and to analyze the samples taken at different fermentation stages by culture-dependent and -independent methods. Three hundred and thirty-eight yeast strains were isolated and twelve genera were identified by sequencing. During the spontaneous fermentation process, non-Saccharomyces yeasts were predominant in the initial and middle stages, whereas Saccharomyces dominated in the later stages; Candida was preponderant in the whole process, and its abundance in the final stages was only lower than Saccharomyces. The inoculated fermentation was characterized by a predominance of Saccharomyces throughout the fermentation process; non-Saccharomyces yeasts were observed in the early stage. The internal transcribed spacer (ITS) 2 region gene was firstly used to analyze the yeast diversity in the samples during the icewine fermentation processes by high-throughput sequencing (HTS), and a more complex mycobiota was revealed. Moreover, the dynamics of other major fungi (mainly Davidiella and Alternaria) during icewine fermentation processes were also revealed, which have never been reported in icewine before.

In this study, the fungal dynamics associated with black locust (BL) mineralization and its correlation with various environmental factors were evaluated across three different vegetation types along a gradient of temperature and humidity. The results confirmed that Ascomycota and Basidiomycota were the dominant phyla in each habitat, with average relative abundance of 86.57 and 11.42%, respectively. But both phylum abundance varied significantly among different BL leaves' decomposing habitats. Black locust changed the most significantly in the forest habitat and the least in the steppe. In addition, the litter characteristics of BL decreased with total carbon and total nitrogen mineralization and underground water level in water-rich region, while this result was significantly consistent with the fungal diversity. Co-occurrence network studies revealed that significant correlations were found between fungal community composition and environmental factors, the decrease of underground water level influence the fungal structure in forest habitat. Finally, the present study results provide important insights about the biological invasion of new ecosystems.