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Norovirus (NoV) is a leading cause of epidemic acute non-bacterial gastroenteritis, and replicates through virion protein genome-linked (VPg)-primed or de novo RNA synthesis by RNA-dependent RNA polymerase (RdRp). VPg is a multifunctional protein that plays crucial roles in viral protein translation and genome replication. However, the interaction between the RdRp and this multifunctional VPg in NoV replication has been unknown. In this study, VPg derived from murine NoV (MNV) was found to mediate the formation of higher-order multimers or tubular fibrils of MNV RdRp, which led to significantly enhanced polymerase activity in vitro. The replication of MNV mutants containing a VPg-binding defective RdRp, based on the crystal structure of an RdRp-VPg(1-73) complex, was completely blocked in a cell culture system. Our data suggest that the interaction between RdRp and VPg plays a crucial role in the multimerization-mediated RdRp activity in vivo and consequently in MNV replication, which can provide a new target of therapeutic intervention for NoV outbreaks.

Transcriptomic and proteomic analysis were performed on 72 h biofilms of the acneic strain Cutibacterium acnes and planktonic cultures in the presence of epinephrine. Epinephrine predominantly downregulated genes associated with various transporter proteins. No correlation was found between proteomic and transcriptomic profiles. In control samples, the expression of 51 proteins differed between planktonic cultures and biofilms. Addition of 5 nM epinephrine reduced this number, and in the presence of 5 µM epinephrine, the difference in proteomic profiles between planktonic cultures and biofilms disappeared. According to the proteomic profiling, epinephrine itself was more effective in the case of C. acnes biofilms and potentially affected the tricarboxylic acid cycle (as well as alpha-ketoglutarate decarboxylase Kgd), biotin synthesis, cell division, and transport of different compounds in C. acnes cells. These findings are consistent with recent research on Micrococcus luteus, suggesting that the effects of epinephrine on actinobacteria may be universal.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) infections are often difficult to treat because of their biofilm-forming ability and antimicrobial resistance. We investigated the effects of sub-minimal inhibitory concentrations (MICs) of antibiotics on MRSA biofilm formation. Clinical MRSA isolates were grown with sub-MICs (1/256–1/2 × MICs) of nafcillin, vancomycin, ciprofloxacin, and rifampin. The biofilm biomass was measured using crystal violet staining. Of the 107 MRSA isolates tested, 63 (58.9%) belonged to sequence type 5 (ST5), and 44 (41.1%) belonged to ST72. The MIC50/MIC90 values of nafcillin, vancomycin, ciprofloxacin, and rifampin were 256/512, 1/2, 64/512, and 0.008/0.03 mg/L, respectively. The sub-MICs of nafcillin, vancomycin, ciprofloxacin, and rifampin promoted biofilm formation in 75 (70.1%), 49 (45.8%), 89 (83.2%), and 89 (83.2%) isolates, respectively. At sub-MICs of nafcillin, the factors associated with strong biofilm induction were the ST5 strain (P = 0.001) and agr dysfunction (P = 0.005). For the sub-MICs of ciprofloxacin, the associated factors were the ST5 strain (P = 0.002), staphylococcal protein A type t002 strain (P < 0.001), and ciprofloxacin resistance (P < 0.001). Among the sub-MICs of rifampin, only ST5 was associated with strong biofilm induction (P = 0.006). Because the sub-MICs of rifampin were much lower than clinically relevant concentrations, we further tested the capability of biofilm induction in 0.03[Formula: see text]32 mg/L of rifampin. At these concentrations, rifampin-induced biofilm formation was rare in rifampin-susceptible MRSA [1.0% (1 of 100)] but common in rifampin-resistant MRSA [71.4% (5 of 7), P < 0.001]. Induction of biofilm biomass at sub-MICs of antibiotics is common in clinical MRSA isolates and is differentially affected by the MRSA strain and antibiotic class.IMPORTANCEBacteria can be exposed to sub-MICs of antibiotics at the beginning and end of a dosing regimen, between doses, or during low-dose therapies. Growing evidence suggests that sub-MICs of antimicrobials can stimulate MRSA biofilm formation and alter the composition of the biofilm matrix. Pevious studies have found that sub-MICs of oxacillin, methicillin, and amoxicillin promote biofilm formation in some community-acquired MRSA (CA-MRSA). We evaluated biofilm induction by sub-MICs of four different classes of antibiotics in 44 CA-MRSA and 63 healthcare-associated MRSA (HA-MRSA) strains. Our study indicated that sub-MICs of nafcillin, vancomycin, ciprofloxacin, and rifampin frequently promote biofilm induction in clinical MRSA isolates. Strong biofilm induction in sub-MICs of nafcillin, ciprofloxacin, and rifampin was more frequent in HA-MRSA than in CA-MRSA. Antibiotic-induced biofilm formation depends on the antibiotic class, MRSA strain, and antibiotic resistance. Our results emphasize the importance of maintaining effective bactericidal concentrations of antibiotics to treat biofilm-related infections.

Background: Expansion of the single sequence type 3 (ST3) was associated with a high macrolide resistance rate among Mycoplasma pneumoniae in Korea during the 2014–2016 epidemic. This study investigates the macrolide resistance rate and genetic diversity of the subsequent epidemic of M. pneumoniae pneumonia in 2019–2020. Methods: The culture for M. pneumoniae was developed from 1228 respiratory samples collected from children with pneumonia in four hospitals in Korea between January 2019 and January 2020. Determination of macrolide resistance and multilocus sequence typing analysis were performed on M. pneumoniae isolates. eBURST analysis was applied to estimate the relationships among strains and to assign strains to a clonal complex. Results: M. pneumoniae was cultured in 93 (7.6%) of 1228 clinical samples. The overall macrolide resistance rate of M. pneumoniae strains was 78.5% (73/93). Of the nine STs identified, three were novel. The most common ST was ST3 (66 [71.0%]) followed by ST14 (18 [19.4%]) and ST7/ST15 (2 [2.2%] each). Three STs (ST3, ST14, and ST17) exhibited macrolide resistance. The macrolide resistance rates of ST3 and ST14 were 98.5% (65 of 66) and 38.9% (7 of 18), respectively. Conclusion: Compared to the previous outbreak in 2014–2016, the overall macrolide resistance remained high; however, an increasing proportion of macrolide resistance was observed within ST14 strains in 2019–2020.

ABSTRACT: Objectives: This study aimed to identify the status of antimicrobial stewardship programmes (ASPs) in small to medium-sized Korean hospitals as well as the awareness and demands about ASPs of physicians. Methods: A questionnaire was designed based on a questionnaire from a previous nationwide survey in 2018 targeting large hospitals in Korea and modified to reflect the results of in-depth interviews with non-infectious diseases (IDs) physicians at secondary care hospitals. The survey targeted all hospitals with ≥150 beds in South Korea and was performed in May–June 2020. Only one ASP-associated physician per hospital participated in the survey. Results: The survey response rate was 31.9% (217/680). ID specialists comprised the majority of medical personnel participating in ASPs in tertiary care hospitals. Conversely, in secondary and primary care hospitals there was no predominant medical personnel for ASPs and the median full-time equivalent was 0 for all types of medical personnel. Tertiary care hospitals, more than secondary and primary care hospitals, tended to perform ASP activities more actively. ‘Workforce for ASPs’, ‘Establishment of healthcare fees for ASPs’ and ‘Development of tools for ASPs’ were the most important required support for ASP improvement. Conclusion: The level of ASP establishment was more limited in primary care hospitals than in secondary and tertiary care hospitals in Korea. To improve ASPs in Korean hospitals, a supporting workforce and the establishment of a healthcare fee for ASPs appear to be necessary.

Abstract Purpose There has been an interest in the microbial azo dye degradation as an optional method for the treatment of azo dye-containing wastes. Tattoo ink is an extremely unique azo dye-rich environment, which have never been explored in terms of microorganisms capable of degrading azo dyes. Previously, we isolated 81 phylogenetically diverse bacteria, belonging to 18 genera and 52 species, contaminated in tattoo inks. In this study, we investigated if these bacteria, which can survive in the azo dye-rich environment, have an ability to degrade azo dyes. Methods We conducted a two-step azo dye degradation (or decolorization) assay. In step 1, a high-throughput degradability assay was done for 79 bacterial isolates using Methyl Red and Orange II. In step 2, a further degradation assay was done for 10 selected bacteria with a representative of 11 azo dyes, including 3 commercial tattoo ink azo dyes. Degradation of azo dyes were calculated from measuring optical absorbance of soluble dyes at specific wavelengths. Results The initial high-throughput azo dye assay (step 1) showed that 79 isolates had a complete or partial degradation of azo dyes; > 90% of Methyl Red and Orange II were degraded within 24 h, by 74 and 20 isolates, respectively. A further evaluation of azo dye degradability for 10 selected isolates in step 2 showed that the isolates, belonging to Bacillus, Brevibacillus, Paenibacillus, and Pseudomonas, exhibited an excellent decolorization ability for a wide range of azo dyes. Conclusions This study showed that phylogenetically diverse bacteria, isolated from azo dye-rich tattoo inks, is able to degrade a diverse range of azo dyes, including 3 azo dyes used in commercial tattoo inks. Some of the strains would be good candidates for future studies to provide a systematic understanding of azo dye degradation mechanisms.

Cases of laboratory-confirmed SARS-CoV-2 reinfection have been reported in a number of countries. Further, the level of natural immunity induced by SARS-CoV-2 infection is not fully clear, nor is it clear if a primary infection is protective against reinfection. To investigate the potential association between serum antibody titres and reinfection of SARS-CoV-2, ferrets with different levels of NAb titres after primary SARS-CoV-2 infection were subjected to reinfection with a heterologous SARS-CoV-2 strain. All heterologous SARS-CoV-2 reinfected ferrets showed active virus replication in the upper respiratory and gastro-intestinal tracts. However, the high NAb titre group showed attenuated viral replication and rapid viral clearance. In addition, direct-contact transmission was observed only from reinfected ferrets with low NAb titres (

SARS-CoV-2 is the third pathogenic coronavirus to emerge since 2000. Experience from prior outbreaks of SARS-CoV and MERS-CoV has demonstrated the importance of both humoral and cellular immunity to clinical outcome, precepts that have been recapitulated for SARS-CoV-2. Despite the unprecedented rapid development and deployment of vaccines against SARS-CoV-2, more vaccines are needed to meet global demand and to guard against immune evasion by newly emerging SARS-CoV-2 variants. Here we describe the development of pGO-1002, a novel bi-cistronic synthetic DNA vaccine that encodes consensus sequences of two SARS-CoV-2 antigens, Spike and ORF3a. Mice immunized with pGO-1002 developed humoral and cellular responses to both antigens, including antibodies and capable of neutralizing infection by a clinical SARS-CoV-2 isolate. Rats immunized with pGO-1002 by intradermal (ID) injection followed by application of suction with our GeneDerm device also developed humoral responses that included neutralizing antibodies and RBD-ACE2 blocking antibodies as well as robust cellular responses to both antigens. Significantly, in a Syrian hamster vaccination and challenge model, ID+GeneDerm-assisted vaccination prevented viral replication in the lungs and significantly reduced viral replication in the nares of hamsters challenged with either an ancestral SARS-CoV-2 strain or the B.1.351 (Beta) variant of concern. Furthermore, vaccinated immune sera inhibited virus-mediated cytopathic effects in vitro. These data establish the immunogenicity of the SARS-CoV-2 vaccine candidate pGO-1002 which induces potent humoral and cellular responses to the Spike and ORF3a antigens and may provide greater protection against emerging variants.

ABSTRACTAs another wave of COVID-19 outbreak has approached in July 2020, a larger scale COVID-19 pediatric Asian cohort summarizing the clinical observations is warranted. Children confirmed with COVID-19 infection from the Republic of Korea, the Hong Kong Special Administrative Region (HKSAR) and Wuhan, China, during their first waves of local outbreaks were included. Their clinical characteristics and the temporal sequences of the first waves of local paediatric outbreaks were compared. Four hundred and twenty three children with COVID-19 were analyzed. Wuhan had the earliest peak, followed by Korea and HKSAR. Compared with Korea and Wuhan, patients in HKSAR were significantly older (mean age: 12.9 vs. 10.8 vs. 6.6 years, p

Noroviruses (NVs) are a major cause of foodborne diseases worldwide. The rhizomes of Acorus gramineus (AGR) have been used as a traditional medicinal plant and a food additive. In this study, AGR and its bioactive components—α-asarone and β-asarone—showed significant antiviral activities against murine NV (MNV) with pre-treatment, with more than two log reductions in viral plaques. They also demonstrated strong inhibition on binding to A- and O-type saliva by the recombinant P domain derived from human NV (HuNV) GII.4. Both α- and β-asarones also inhibited the binding of the P domain to the receptor at 0.125–1 mM in a concentration-dependent manner and induced a marked reduction in Tm, suggesting that they may reduce structural stability and block receptor binding by the P domain. In simulated digestive conditions, the AGR extract, α-asarone, or β-asarone further showed a significant reduction of MNV plaques by 1.5–2.8 logs. The asarones show a potential for development as a scaffold for anti-NV agents.

The inhalation toxicity of carbon nanofibers (CNFs) is not clearly known due to relatively few related studies reported. An acute inhalation study and short-term inhalation study (5 days) were therefore conducted using Sprague-Dawley rats. In the acute inhalation study, the rats were grouped and exposed to a fresh air control or to low (0.238 ± 0.197), moderate (1.935 ± 0.159), or high (24.696 ± 6.336 mg/m3) CNF concentrations for 6 h and thereafter sacrificed at 14 days. For the short-term inhalation study, the rats were grouped and exposed to a fresh air control or low (0.593 ± 0.019), moderate (2.487 ± 0.213), or high (10.345 ± 0.541 mg/m3) CNF concentrations for 6 h/day for 5 days and sacrificed at 1, 3, and 21 days post-exposure. No mortality was observed in the acute inhalation study. Thus, the CNF LC50 was higher than 25 mg/m3. No significant body or organ weight changes were noted during the 5 days short-term inhalation study or during the post-exposure period. No significant effects of toxicological importance were observed in the hematological, blood biochemical, and coagulation tests. In addition, the bronchoalveolar lavage (BAL) fluid cell differential counts and BAL inflammatory markers showed no CNF-exposure-relevant changes. The histopathological examination also found no CNF-exposure-relevant histopathological lesions. Thus, neither acute nor 5 days inhalation exposure to CNFs induced any noticeable toxicological responses.

The recent dramatic decline in sea ice conditions in the Arctic Ocean has led to the ecophysiological changes in the phytoplankton community. Little is currently known about how the physiological status of phytoplankton has changed under rapidly changing environmental conditions in the Arctic Ocean. Using the 13C isotope tracer technique, the carbon allocation of phytoplankton into different photosynthetic end-products was determined in the northern Chukchi Sea on the basis of two Arctic expeditions conducted in 2011 and 2012 to identify the physiological status of phytoplankton. Lipids were the predominant photosynthetic biochemical fraction (42.5%) in 2011, whereas carbon allocation to proteins was most dominant under ice-free conditions in 2012 (47.7%). Based on a comparison of the photosynthetic carbon allocation of phytoplankton according to sea ice conditions, we found that photosynthetic carbon allocation to different macromolecular pools was significantly different depending on the sea ice conditions and that the light conditions caused by different sea ice conditions could be an important reason for the differences in carbon allocation to photosynthetic end-products. Different dominant phytoplankton groups related to size classes also could cause changes in the photosynthetic carbon allocation of phytoplankton related mainly to the lipid synthesis. Our results showed that the physiological status of Arctic phytoplankton could be changed by producing different photosynthetic end-products under current environmental changes. This change in photosynthetic end-products of phytoplankton as a basic food source could be further linked to higher trophic levels in regards to their nutritional and energetic aspects, which could have potential consequences for Arctic marine ecosystems.

Norovirus is the leading cause of nonbacterial foodborne disease outbreaks. Human noroviruses (HuNoVs) bind to histo-blood group antigens as the host receptor for infection. In this study, the inhibitory effects of fucoidans from brown algae, Laminaria japonica (LJ), Undaria pinnatifida and Undaria pinnatifida sporophyll, were evaluated against murine norovirus (MNoV), feline calicivirus (FCV) and HuNoV. Pretreatment of MNoV or FCV with the fucoidans at 1 mg/mL showed high antiviral activities, with 1.1 average log reductions of viral titers in plaque assays. They also showed significant inhibition on the binding of the P domains of HuNoV GII.4 and GII.17 to A- or O-type saliva and the LJ fucoidan was the most effective, reaching 54–72% inhibition at 1 mg/mL. In STAT1−/− mice infected with MNoV, oral administration of the LJ fucoidan, composed of mainly sulfated fucose and minor amounts of glucose and galactose, improved the survival rates of mice and significantly reduced the viral titers in their feces. Overall, these results provide the LJ fucoidan can be used to reduce NoV outbreaks.

The ubiquitin–proteasome system (UPS) is an essential protagonist in host–pathogen interactions. Among the three classes of enzymes in the UPS, ubiquitin-conjugating enzyme E2 plays a dual role in viral pathogenesis; however, the role of insect E2s in interactions with plant viruses is unclear. Twenty E2-encoding genes in Laodelphax striatellus, the small brown planthopper, were identified and classified into 17 groups by transcriptomic and phylogenetic analysis. Full-length cDNAs of four LstrE2s (LstrE2 A/E/G2/H) were obtained by rapid-amplification of cDNA ends (RACE-PCR) analysis. Expression of the four LstrE2s showed tissue- and development-specific patterns. RT-qPCR analyses revealed that Rice stripe viruse (RSV) infection increased the level of LstrE2 A/E/G2/H. Further study indicated that repression of LstrE2 E via RNAi caused significant increases in the expression of RSV coat protein mRNA and protein levels. These findings suggest that LstrE2 E inhibits RSV accumulation in the planthopper body. Understanding the function of LstrE2 E in RSV accumulation may ultimately result in the development of novel antiviral strategies.

Baicalein (3,3′,4′,5,6-pentahydroxyflavone) is a well-known antioxidant found in many plants, such as in the roots of Scutellaria baicalensis. In this study, we evaluate the inhibitory effect of baicalein on the inflammatory cascade in RAW 264.7 mouse macrophages induced by viral-like material. Experimental assays used in this study included Griess reagent assay for nitric oxide (NO) production, Fluo-4 assay for intracellular calcium release, multiplex cytokine assay, and quantitative real time RT-PCR assay. To induce inflammation, RAW 264.7 cells were treated with polyinosinic–polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA). Baicalein at concentrations up to 100 μM significantly inhibited the production of NO, IL-1α, IL-6, G-CSF, GM-CSF, VEGF, MCP-1, IP-10, LIX, and RANTES as well as calcium release in RAW 264.7 cells induced by poly I:C (50 µg/mL) (all p < 0.05). Baicalein at concentrations up to 50 μM also significantly inhibited mRNA expression of STAT1, STAT3, CHOP, and Fas in poly I:C-induced RAW 264.7 cells (p < 0.05). In conclusion, baicalein has anti-inflammatory effect in double-stranded RNA (dsRNA)-induced macrophages by inhibiting NO, cytokines, chemokines, and growth factors via the endoplasmic reticulum stress–CHOP/STAT pathway.

Root colonization by Pseudomonas chlororaphis O6 in cucumber elicited an induced systemic resistance (ISR) against Corynespora cassiicola. In order to gain insight into O6-mediated ISR, a suppressive subtractive hybridization technique was applied and resulted in the isolation of a cucumber galactinol synthase (CsGolS1) gene. The transcriptional level of CsGolS1 and the resultant galactinol content showed an increase several hours earlier under O6 treatment than in the water control plants following C. cassiicola challenge, whereas no difference was detected in the plants without a pathogen challenge. The CsGolS1-overexpressing transgenic tobacco plants demonstrated constitutive resistance against the pathogens Botrytis cinerea and Erwinia carotovora, and they also showed an increased accumulation in galactinol content. Pharmaceutical application of galactinol enhanced the resistance against pathogen infection and stimulated the accumulation of defense-related gene transcripts such as PR1a, PR1b, and NtACS1 in wild-type tobacco plants. Both the CsGolS1-overexpressing transgenic plants and the galactinol-treated wild-type tobacco plants also demonstrated an increased tolerance to drought and high salinity stresses.

Caliciviruses are a leading agent of human and animal gastroenteritis and respiratory tract infections, which are growing concerns in immunocompromised individuals. However, no vaccines or therapeutics are yet available. Since the rapid rate of genetic evolution of caliciviruses is mainly due to the error-prone nature of RNA-dependent RNA polymerase (RdRp), this article focuses on recent studies of the structures and functions of RdRp from caliciviruses. It also provides recent advances in the interactions of RdRp with virion protein genome-linked (VPg) and RNA and the structural and functional features of its precursor.

Influenza is a serious public health concern worldwide, as it causes significant morbidity and mortality. The emergence of drug-resistant viral strains requires new approaches for the treatment of influenza. In this study, Rubus coreanus seed (RCS) that is left over from the production of wine or juice was found to show antiviral activities against influenza type A and B viruses. Using the time-of-addition plaque assay, viral replication was almost completely abolished by simultaneous treatment with the RCS fraction of less than a 1-kDa molecular weight (RCSF1). One of the polyphenols derived from RCSF1, gallic acid (GA), identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against both influenza type A and B viruses, albeit at relatively high concentrations. RCSF1 was bound to hemagglutinin protein, inhibited hemagglutination significantly and disrupted viral particles, whereas GA was found to only disrupt the viral particles by using transmission electron microscopy. In BALB/c mice infected with influenza virus, oral administration of RCSF1 significantly improved the survival rate and reduced the viral titers in the lungs. Our results demonstrate that RCSF1 and GA show potent and broad antiviral activity against influenza A and B type viruses and are promising sources of agents that target virus particles.