Monitoring viruses and beta-lactam resistance genes through wastewater surveillance during a COVID-19 surge in Suwon, South Korea.

The present study reports data on a long-term campaign for monitoring SARS-CoV-2, norovirus, hepatitis A virus, and beta-lactam resistance genes in wastewater samples from a wastewater treatment plant during COVID-19 surge in Suwon, South Korea. Real-time digital PCR (RT-dPCR) assays indicated 100 % occurrence of all but hepatitis A virus and blaNDM gene in influent wastewater samples. CDC-N1 assay detected SARS-CoV-2 in all influent samples with an average log-transformed concentration of 5.1 ± 0.39 and the highest level at 6.02 gene copies/L. All samples were also positive for norovirus throughout the study with a mean concentration 5.67 ± 0.65 log10 gene copies/L. On the contrary, all treated wastewater (effluent) tested negative for both viruses' genetic materials. Furthermore, plasmid-mediated AmpC ß-lactamases (PABLs) genes blaDHA, blaACC, and blaFOX, extended-spectrum ß-lactamases (ESBLs) genes blaTEM and blaCTX, and Klebsiella pneumoniae carbapenemase (blaKPC) gene were measured at average concentrations of 7.05 ± 0.26, 5.60 ± 0.35, 7.82 ± 0.43, 8.38 ± 0.20, 7.64 ± 0.29, and 7.62 ± 0.41 log10 gene copies/L wastewater, respectively. Beta-lactam resistance genes showed strong correlations (r), the highest being 0.86 for blaKPC - blaFOX, followed by 0.82 for blaTEM - blaCTX and 0.79 for blaTEM - blaDHA. SARS-CoV-2 RNA occurrence in the wastewater was strongly associated (r = 0.796) with COVID-19 cases in the catchment during the initial study period of six months. A positive association of the SARS-CoV-2 RNA with the prevalence of COVID-19 cases showed a promising role of community-scale monitoring of pathogens to provide considerable early signals of infection dynamics. High concentrations of beta-lactam resistance genes in wastewater indicated a high concern for one of the biggest global health threats in South Korea and the need to find control measures. Moreover, antibiotic-resistance genes in treated wastewater flowing through water bodies and agricultural environments indicate further dissemination of antibiotic resistance traits and increasing microbial antibiotic resistance.

Fibrivirga algicola gen. nov., sp. nov., an algicidal bacterium isolated from a freshwater river.

An aerobic, gram-stain-negative, pink-colored, non-motile and rod-shaped algicidal bacterium, designated as JA-25T was isolated from freshwater in Geumgang River, Republic of Korea. Strain JA-25T grew at 15-30 °C and pH 6-9, and did not require NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain JA-25T belongs to the family 'Spirosomaceae' and is most closely related to Fibrella aestuarina BUZ 2T (93.6%). Strain JA-25T showed < 90% sequence similarity to other members of the family 'Spirosomaceae'. The average nucleotide identity(ANI), in silico DNA-DNA hybridization and average amino acid identity(AAI) values based on the genomic sequences of JA-25T and F. aestuarina BUZ 2T were 74.4, 20.5, and 73.6%, respectively. Strain JA-25T showed an algicidal effect on the marine flagellate alga Heterocapsa triquetra, but no effect on fresh water cyanobacterium (Nostoc). In genome analysis, RIPP-like peptides were detected and predicted to resemble the indolmycin biosynthetic gene cluster, which possibly influence its algicidal effect. Furthermore, a bacteriorhodopsin gene with photoheterotrophic characteristics was detected. The genomic DNA G + C content was 52.5 mol%. The major cellular fatty acids were summed feature 3 (C16:1 ω6c/C16:1 ω7c), C16:1 ω5c, C16:0 (> 10%). The major respiratory quinone was menaquinone 7 and major polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two phospholipids, and five unidentified lipids. Considering the phylogenetic inference, phenotypic, and chemotaxonomic data, strain JA-25T should be classified as a novel species in the novel genus Fibrivirga, with the proposed name Fibrivirga algicola sp. nov. The type strain is JA-25T (= KCCM 43334T = NBRC 114259T).

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

Achieving Maximal Production of Fusaricidins from Paenibacillus kribbensis CU01 via Continuous Fermentation.

In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l-1) was achieved in a modified M9 medium containing metal ions, 10 g l-1 glucose, and 1 g l-1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l-1 h-1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l-1 glucose, 1 × 10-3 M FeSO4, and 1 × 10-6 M MnCl2. After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min-1, and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l-1) was obtained using a dilution rate of 0.075 h-1 with an average productivity of 10.4 mg l-1 h-1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

Seeking Consensus on the Terminology of Value-Based Transformation Through use of a Delphi Process.

Collaboration among diverse stakeholders involved in the value transformation of health care requires consistent use of terminology. The objective of this study was to reach consensus definitions for the terms value-based care, value-based payment, and population health. A modified Delphi process was conducted from February 2017 to July 2017. An in-person panel meeting was followed by 3 rounds of surveys. Panelists anonymously rated individual components of definitions and full definitions on a 9-point Likert scale. Definitions were modified in an iterative process based on results of each survey round. Participants were a panel of 18 national leaders representing population health, health care delivery, academic medicine, payers, patient advocacy, and health care foundations. Main measures were survey ratings of definition components and definitions. At the conclusion of round 3, consensus was reached on the following definition for value-based payment, with 13 of 18 panelists (72.2%) assigning a high rating (7- 9) and 1 of 18 (5.6%) assigning a low rating (1-3): "Value-based payment aligns reimbursement with achievement of value-based care (health outcomes/cost) in a defined population with providers held accountable for achieving financial goals and health outcomes. Value-based payment encourages optimal care delivery, including coordination across healthcare disciplines and between the health care system and community resources, to improve health outcomes, for both individuals and populations." The iterative process elucidated specific areas of agreement and disagreement for value-based care and population health but did not reach consensus. Policy makers cannot assume uniform interpretation of other concepts underlying health care reform efforts.

Microbial consortia including methanotrophs: some benefits of living together.

With the progress of biotechnological research and improvements made in bioprocessing with pure cultures, microbial consortia have gained recognition for accomplishing biological processes with improved effectiveness. Microbes are indispensable tool in developing bioprocesses for the production of bioenergy and biochemicals while utilizing renewable resources due to technical, economic and environmental advantages. They communicate with specific cohorts in close proximity to promote metabolic cooperation. Use of positive microbial associations has been recognized widely, especially in food industries and bioremediation of toxic compounds and waste materials. Role of microbial associations in developing sustainable energy sources and substitutes for conventional fuels is highly promising with many commercial prospects. Detoxification of chemical contaminants sourced from domestic, agricultural and industrial wastes has also been achieved through microbial catalysis in pure and co-culture systems. Methanotrophs, the sole biological sink of greenhouse gas methane, catalyze the methane monooxygenasemediated oxidation of methane to methanol, a high energy density liquid and key platform chemical to produce commodity chemical compounds and their derivatives. Constructed microbial consortia have positive effects, such as improved biomass, biocatalytic potential, stability etc. In a methanotroph-heterotroph consortium, non-methanotrophs provide key nutrient factors and alleviate the toxicity from the culture. Non-methanotrophic organisms biologically stimulate the growth and activity of methanotrophs via production of growth stimulators. However, methanotrophs in association with co-cultured microorganisms are in need of further exploration and thorough investigation to study their interaction mode and application with improved effectiveness.

Altererythrobacter rubellus sp. nov., a marine alphaproteobacterium isolated from seawater.

The phylogenetic and taxonomic status of an alphaproteobacterium isolated from seawater, collected in the Republic of Korea, was elucidated based on a polyphasic method. Strain KMU-45T was Gram-stain-negative, strictly aerobic, rod-shaped, non-motile and chemoheterotrophic. Phylogenetic investigation based on the 16S rRNA gene sequence demonstrated that the novel marine isolate belongs to the family Erythrobacteraceae, of the class Alphaproteobacteria, and that it possessed the highest (98.7%) sequence similarity with Altererythrobacter ishigakiensis ATCC BAA-2084T. DNA-DNA hybridization values between strains KMU-45T and A. ishigakiensis KCTC 42446T were 31.4% ± 8.4%. The major isoprenoid quinone of the novel isolate was ubiquinone-10 (Q-10) and the major (>10%) cellular fatty acids were C17:1 ω8c, C17:1 ω6c and C18:1 ω7c. The genomic DNA G + C content of strain KMU-45T was 57.7 mol%. The polar lipid profile of the strain KMU-45T comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified phospholipid and an unidentified lipid. From the polyphasic taxonomic results in this study, the bacterium can be considered to represent a new species of the genus Altererythrobacter, for which the name Altererythrobacter rubellus sp. nov. is proposed. The type species is A. rubellus sp. nov., with the type strain KMU-45T (= KCCM 90270T = NBRC 112769T).

Expression Profile of Three Splicing Factors in Pleural Cells Based on the Underlying Etiology and Its Clinical Values in Patients with Pleural Effusion.

Splicing factors (SFs) are involved in oncogenesis or immune modulation, the common underlying processes giving rise to pleural effusion (PE). The expression profiles of three SFs (HNRNPA1, SRSF1, and SRSF3) and their clinical values have never been assessed in PE. The three SFs (in pellets of PE) and conventional tumor markers were analyzed using PE samples in patients with PE (N = 336). The sum of higher-molecular weight (Mw) forms of HNRNPA1 (Sum-HMws-HNRNPA1) and SRSF1 (Sum-HMws-SRSF1) and SRSF3 levels were upregulated in malignant PE (MPE) compared to benign PE (BPE); they were highest in cytology-positive MPE, followed by tuberculous PE and parapneumonic PE. Meanwhile, the lowest-Mw HNRNPA1 (LMw-HNRNPA1) and SRSF1 (LMw-SRSF1) levels were not upregulated in MPE. Sum-HMws-HNRNPA1, Sum-HMws-SRSF1, and SRSF3, but neither LMw-HNRNPA1 nor LMw-SRSF1, showed positive correlations with cancer cell percentages in MPE. The detection accuracy for MPE was high in the order of carcinoembryonic antigen (CEA, 85%), Sum-HMws-HNRNPA1 (76%), Sum-HMws-SRSF1 (68%), SRSF3, cytokeratin-19 fragments (CYFRA 21-1), LMw-HNRNPA1, and LMw-SRSF1. Sum-HMws-HNRNPA1 detected more than half of the MPE cases that were undetected by cytology and CEA. Sum-HMws-HNRNPA1, but not other SFs or conventional tumor markers, showed an association with longer overall survival among patients with MPE receiving chemotherapy. Our results demonstrated different levels of the three SFs with their Mw-specific profiles depending on the etiology of PE. We suggest that Sum-HMws-HNRNPA1 is a supplementary diagnostic marker for MPE and a favorable prognostic indicator for patients with MPE receiving chemotherapy.

Structural analysis and enhanced production of fusaricidin from Paenibacillus kribbensis CU01 isolated from yellow loess.

A bacterial strain showing strong antifungal activity was isolated from yellow loess and was identified as Paenibacillus kribbensis CU01. Insoluble mucoidal polymers were separated from M9 culture medium via low-speed centrifugation. Most antifungal activity was associated with substances in the insoluble precipitate, which was purified by reverse phase high performance liquid chromatography. Purified fractions were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Two major ion peaks with mass-to-charge ratio values (m/z) at 883.6 and 897.6 were revealed. After alkaline hydrolysis and sequence analysis, two cyclic depsipeptides were identified as, fusaricidin A and fusaricidin B. Their production was significantly increased by the addition of glucose, Fe2+ , and Mn2+ to M9 medium. Maximum concentrations of produced fusaricidin A and fusaricidin B at flask-scale comprised 460 mg L-1 and 118 mg L-1 , respectively: the highest production concentrations yet reported in the literature. This demonstrates that P. kribbensis CU01 has enormous commercial potential for the mass production of fusaricidin.

HNRNPA1, a Splicing Regulator, Is an Effective Target Protein for Cervical Cancer Detection: Comparison With Conventional Tumor Markers.

OBJECTIVE: Heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), serine/arginine-rich splicing factor 1 (SRSF1), and SRSF3 are splicing regulators associated with oncogenesis. However, the alterations of SF proteins and their diagnostic values in cervical cancer are unclear. To apply SFs clinically, effective marker selection and characterization of the target organ properties are essential. MATERIALS AND METHODS: We concurrently analyzed HNRNPA1, SRSF1, SRSF3, and the conventional tumor markers squamous cell carcinoma antigen (SCCA) and carcinoembryonic antigen (CEA) in cervical tissue samples (n = 127) using semiquantitative immunoblotting. In addition, we compared them with p16 (cyclin-dependent kinase inhibitor 2A [CDKN2A]), which has shown high diagnostic efficacy in immunohistochemical staining studies and has been proposed as a candidate protein for point-of-care screening biochemical tests of cervical neoplasia. RESULTS: HNRNPA1, higher molecular weight forms of SRSF1 (SRSF1-HMws), SRSF3, CEA, and p16 levels were higher (P < 0.05) in cervical carcinoma tissue samples than in nontumoral cervical tissue samples. However, the levels of SRSF1-Total (sum of SRSF1-HMws and a lower molecular weight form of SRSF1) and SCCA, a commonly used cervical tumor marker, were not different between carcinoma and nontumoral tissue samples. In paired sample comparisons, HNRNPA1 (94%) showed the highest incidence of up-regulation (carcinoma/nontumor, >1.5) in cervical carcinoma, followed by p16 (84%), SRSF1-HMws (69%), SRSF3 (66%), CEA (66 %), SCCA (32%), and SRSF1-Total (31%). HNRNPA1 (92%) and p16 (91%) presented the two highest diagnostic accuracies for cervical carcinoma, which were superior to those of SRSF3 (75%), SRSF1-HMws (72%), CEA (72%), SCCA (59%), and SRSF1-Total (55%). CONCLUSIONS: Our results identified that HNRNPA1 is the best diagnostic marker among the SFs and conventional markers given its excellent diagnostic efficacy for cervical carcinoma, and it has a p16-comparable diagnostic value. We suggest that HNRNPA1 is an additional effective target protein for developing cervical cancer detection tools.

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions.

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m(2)/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16-C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.

Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

Effects of the algicide, thiazolidinedione derivative TD49, on microbial communities in a mesocosm experiment.

We investigated the effects of the algicide thiazolidinedione derivative TD49 on microbial community in mesocosm experiments. The TD49 concentration exponentially decreased over time, with half-life of 3.5 h, following addition in the seawater (R2=0.98, P<0.001). Among microbial communities, heterotrophic bacteria and heterotrophic nanoflagellates (HNFs) grew well in all treatments following the addition of TD49. The abundance of HNFs lagged behind the increase in heterotrophic bacteria by 24 h in the 0.2 and 0.4 µM TD49 concentrations (R2=0.28, P<0.05), and by 48 h in the 0.6 and 1.0 µM TD49 concentrations (R2=0.30, P<0.05). This implies a strong concentration-dependent top-down effect of TD49 on microbial communities, with indications that the degradation of planktonic organisms, including the target alga, led to high heterotrophic bacteria concentrations, which in turn stimulated the population growth of predatory HNF. However, total ciliate numbers remained relatively low in the TD49 treatments relative to the control and blank groups, suggesting limited carbon flow from bacteria to these grazers even though the abundance of aloricate ciliates gradually increased toward the end of the experimental period, particularly at the high TD49 concentrations. TD49 appears to provide an environmentally safe approach to the control of harmful algal blooms (HABs) in aquatic ecosystems.

Construction of target-specific virus-like particles for the delivery of algicidal compounds to harmful algae.

Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems.

Quality at Kaiser Permanente: using the population care model.

The provision of high quality healthcare is facilitated by an integrated team of multi-specialty physicians who are supported by an advanced electronic medical record. This paper shows how Kaiser Permanente of the Mid-Atlantic States is able to provide proactive care to members through physicians and their teams, integrated with functional health information technology systems.

Production of biosurfactant lipopeptides Iturin A, fengycin and surfactin A from Bacillus subtilis CMB32 for control of Colletotrichum gloeosporioides.

A bacterial strain isolated from soil for its potential to control the anthracnose disease caused by Colletotrichum gloeosporioides was identified as a Bacillus subtilis. Bacillus subtilis CMB32 produced antifungal agents on M9 broth at 30degreesC. Biosurfactant lipopeptides produced by Bacillus subtilis CMB32 were precipitated by adjusting to pH 2 and extracting using chloroform/methanol, and then were purified using column chromatography and reverse-phase HPLC. Molecular masses of the lipopeptides were estimated by MALDI-TOF mass spectrometry as (a) 1080, (b) 1486, and (c) 1044 Da, respectively. They had cyclic structures and amino acid compositions of (a) Pro, Asx, Ser, Tyr, Glx, (b) Glx, Tyr, Thr, Ala, Pro, Ile, and (c) Glx, Leu, Val, Asx, respectively. Further analysis revealed that Bacillus subtilis CMB32 produced three antifungal lipopeptides: (a) iturin A, (b) fengycin, and (c) surfactin A.