Construction of Bacillus thuringiensis simulant strains suitable for environmental release

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/β-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment

Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate

Short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC) inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP). Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine

Molecular characterization of SARS‐CoV‐2 from the saliva of patients in the Republic of Korea in 2020

Abstract Background and aims Despite global vaccination efforts, the number of confirmed cases of coronavirus disease 2019 (COVID‐19) remains high. To overcome the crisis precipitated by the ongoing pandemic, characteristic studies such as virus diagnosis, isolation, and genome analysis of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are necessary. Herein, we report the isolation and molecular characterization of SARS‐CoV‐2 from the saliva of patients who had tested positive for COVID‐19 at Proving Ground in Taean County, Republic of Korea, in 2020. Methods We analyzed the whole‐genome sequence of SARS‐CoV‐2 isolated from the saliva samples of patients through next‐generation sequencing. We also successfully isolated SARS‐CoV‐2 from the saliva samples of two patients by using cell culture, which was used to study the cytopathic effects and viral replication in Vero E6 cells. Results Whole‐genome sequences of the isolates, SARS‐CoV‐2 ADD‐2 and ADD‐4, obtained from saliva were identical, and phylogenetic analysis using Bayesian inference methods showed SARS‐CoV‐2 GH clade (B.1.497) genome‐specific clustering. Typical coronavirus‐like particles, with diameters of 70–120 nm, were observed in the SARS‐CoV‐2 infected Vero E6 cells using transmission electron microscopy. Conclusion In conclusion, this report provides insights into the molecular diagnosis, isolation, genetic characteristics, and diversity of SARS‐CoV‐2 isolated from the saliva of patients. Further studies are needed to explore and monitor the evolution and characteristics of SARS‐CoV‐2 variants

Respiratory Complications Associated with Insertion of Small-Bore Feeding Tube in Critically Ill Patients

Small-bore flexible feeding tubes decrease the risk of ulceration of the nose, pharynx, and stomach compared with large-bore and more rigid tubes. However, small-bore feeding tubes have more respiratory system complications, such as pneumothorax, hydropneumothorax, bronchopleural fistula, and pneumonia, which are associated with significant morbidity and mortality. Thus, it is important to confirm the correct position of feeding tubes. Chest X-ray is the gold standard to detect tracheal malpositioning of the feeding tube. We present three cases in which intubated patients exhibited an altered mental state. An assistant guide wire was used at the insertion of small-bore feeding tubes. These conditions are thought to be potential risk factors for tracheobronchial malpositioning of feeding tubes

Ischemic Stroke in Critically Ill Patients with Malignancy.

Cerebrovascular diseases are a frequent cause of neurological symptoms in patients with cancer. The clinical characteristics of ischemic stroke (IS) in patients with cancer have been reported in several studies; however, limited data are available regarding critically ill patients with cancer who develop IS during their stay in the intensive care unit (ICU).All consecutive patients who underwent brain magnetic resonance imaging (MRI) for suspicion of IS with acute abnormal neurologic symptoms or who developed signs of IS while in the ICU were retrospectively evaluated. We compared the clinical characteristics and diffusion-weighted imaging (DWI) lesion patterns between patients finally diagnosed as having or not having IS.Over the study period, a total of 88 patients underwent brain MRI for suspicion of IS, with altered mental status in 55 (63%), hemiparesis in 28 (32%), and seizure in 20 (23%). A total of 43 (49%) patients were ultimately diagnosed with IS. Multiple DWI lesions (41%) were more common than single lesions (8%). The etiologies of IS were not determined in the majority of patients (n = 27, 63%). In the remaining 16 (37%) patients, the most common aetiology of IS was cardioembolism (n = 8), followed by large-vessel atherosclerosis (n = 3) and small-vessel occlusion (n = 2). However, brain metastases were newly diagnosed in only 7 (8%) patients. Univariate comparison of the baseline characteristics between patients with or without IS did not reveal any significant differences in sex, malignancy type, recent chemotherapy, vascular risk factors, or serum D-dimer levels at the time of suspicion of IS. Thrombotic events were more common in the IS group than in the non-IS group (P = 0.028). However, patients who were ultimately diagnosed with IS had more hemiparesis symptoms at the time of suspicion of IS (P = 0.001). This association was significant even after adjusting for potentially confounding factors (adjusted odds ratio 5.339; 95% confidence interval, 1.521-19.163).IS developed during ICU stays in critically ill patients with cancer have particular features that may be associated with cancer-related mechanism

Safety and Feasibility of Percutaneous Dilatational Tracheostomy Performed by Intensive Care Trainee

Background: Percutaneous dilatational tracheostomy (PDT) performed by an intensivist in critically ill patients is currently popular. Many studies support the safety and feasibility of PDT. However, there is limited data on the safety and feasibility of PDT performed by intensive care trainees. Methods: To evaluate the safety and feasibility of PDT performed by intensive care trainees and to compare these with those performed by intensivists, we retrospectively analyzed the clinical characteristics and adverse events of all prospectively registered patients who underwent PDT by ICT or intensivists in intensive care units (ICUs) from August 2010 to August 2013. Results: In the study period, 203 patients underwent PDT in ICUs; 139 (68%) by trainees and 64 (32%) by intensivists. There were no statistically significant differences in clinical characteristics including demographics, laboratory findings, and parameters of mechanical ventilation between the two groups. Procedure times and outcomes of the patients were not different between the two groups. The majority of complications observed in 24 hours after PDT were bleeding; however, there was no significant difference between the two groups (trainee 10.8% vs. intensivist 9.4%, p = 0.758). There was no procedure-related death in the two groups. Conclusions: PDT performed by intensive care trainees was safe and feasible. However, further well-designed studies should be conducted to confirm our results

Phylogeographic diversity and hybrid zone of Hantaan orthohantavirus collected in Gangwon Province, Republic of Korea.

BackgroundHantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius (the striped field mouse), causes hemorrhagic fever with renal syndrome (HFRS) in humans. Viral genome-based surveillance at new expansion sites to identify HFRS risks plays a critical role in tracking the infection source of orthohantavirus outbreak. In the Republic of Korea (ROK), most studies demonstrated the serological prevalence and genetic diversity of orthohantaviruses collected from HFRS patients or rodents in Gyeonggi Province. Gangwon Province is a HFRS-endemic area with a high incidence of patients and prevalence of infected rodents, ROK. However, the continued epidemiology and surveillance of orthohantavirus remain to be investigated.Methodology/principal findingsWhole-genome sequencing of HTNV was accomplished in small mammals collected in Gangwon Province during 2015-2018 by multiplex PCR-based next-generation sequencing. To elucidate the geographic distribution and molecular diversity of viruses, we conducted phylogenetic analyses of HTNV tripartite genomes. We inferred the hybrid zone using cline analysis to estimate the geographic contact between two different HTNV lineages in the ROK. The graph incompatibility based reassortment finder performed reassortment analysis. A total of 12 HTNV genome sequences were completely obtained from A. agrarius newly collected in Gangwon Province. The phylogenetic and cline analyses demonstrated the genetic diversity and hybrid zone of HTNV in the ROK. Genetic exchange analysis suggested the possibility of reassortments in Cheorwon-gun, a highly HFRS-endemic area.Conclusions/significanceThe prevalence and distribution of HTNV in HFRS-endemic areas of Gangwon Province enhanced the phylogeographic map for orthohantavirus outbreak monitoring in ROK. This study revealed the hybrid zone reflecting the genetic diversity and evolutionary dynamics of HTNV circulating in Gangwon Province. The results arise awareness of rodent-borne orthohantavirus diseases for physicians in the endemic area of ROK

A Portable Diagnostic Assay, Genetic Diversity, and Isolation of Seoul Virus from <i>Rattus norvegicus</i> Collected in Gangwon Province, Republic of Korea

Seoul virus (SEOV), an etiological agent for hemorrhagic fever with renal syndrome, poses a significant public health threat worldwide. This study evaluated the feasibility of a mobile Biomeme platform for facilitating rapid decision making of SEOV infection. A total of 27 Rattus norvegicus were collected from Seoul Metropolitan City and Gangwon Province in Republic of Korea (ROK), during 2016–2020. The serological and molecular prevalence of SEOV was 5/27 (18.5%) and 2/27 (7.4%), respectively. SEOV RNA was detected in multiple tissues of rodents using the Biomeme device, with differences in Ct values ranging from 0.6 to 2.1 cycles compared to a laboratory benchtop system. Using amplicon-based next-generation sequencing, whole-genome sequences of SEOV were acquired from lung tissues of Rn18-1 and Rn19-5 collected in Gangwon Province. Phylogenetic analysis showed a phylogeographical diversity of rat-borne orthohantavirus collected in Gangwon Province. We report a novel isolate of SEOV Rn19-5 from Gangwon Province. Our findings demonstrated that the Biomeme system can be applied for the molecular diagnosis of SEOV comparably to the laboratory-based platform. Whole-genome sequencing of SEOV revealed the phylogeographical diversity of orthohantavirus in the ROK. This study provides important insights into the field-deployable diagnostic assays and genetic diversity of orthohantaviruses for the rapid response to hantaviral outbreaks in the ROK