Toyama Institute of Health

Abstract Background Epigenetic clocks based on DNA methylation (DNAm) are widely used indicators of biological aging; however, most established models have been developed using EPIC arrays and non-Japanese populations. The Methylation Screening Array (MSA), a cost-efficient platform with reduced CpG content, has not been evaluated for its capacity to support biological age estimation and biomarker prediction in Japanese cohorts. Methods DNAm profiles and clinical laboratory measurements were obtained from 166 Japanese participants for model development; an independent cohort of 48 individuals processed at a separate institute was used for validation. A linear regression model was trained using the Elastic Net method to predict phenotypic age from MSA-derived methylation data, and a two-stage modeling (residual learning) framework integrating EPIC-based clock predictions with MSA-specific residual predictions was evaluated. Additional models were constructed to examine the predictability of 59 clinical biomarkers and their log-transformed variants, including sex-stratified analyses. Results The MSA-based model accurately predicted phenotypic age in the validation dataset; prediction performance improved when the EPIC-based estimates were incorporated through the residual learning framework. Several clinical biomarkers, particularly those related to leukocyte composition and sex hormone regulation, were also predicted from the MSA data, although some markers were strongly affected by sex. Some of the nine constituent phenotypic age biomarkers were not individually predicted. Conclusions MSA methylation profiles contain sufficient biological information for reliable prediction of epigenetic aging markers in Japanese individuals. These findings demonstrate the feasibility of applying cost-efficient MSA-based DNAm profiling for biological age prediction and provide a methodological foundation for expanding epigenetic biomarker applications in Japan.

We developed a convenient and simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method for analysis of iodide in concentrated chloride springs. Using a C18 column, the iodide and chloride peaks were well separated. Setting the wavelength at 240nm allows direct ultraviolet (UV) detection of iodide without any UV or Schlieren effect due to the high chloride concentration. The high ionic strength of spring samples containing high levels of sodium chloride broadens the iodide peak. Iodide was quantified based on the peak areas of standard solutions of iodide, in which 100mM sodium chloride was added to match the saline matrixes with the samples. The detection limit of iodide at a signal-to-noise ratio of 3 was 0.0003mM. The detector response was linear over the concentration ranges of 0.001-0.1mM. The proposed RP-HPLC/UV method was successfully applied to determine iodide in some concentrated chloride spring samples.

Yersinia pseudotuberculosis (Ypt) is a gram-negative bacterium that infects both humans and animals primarily through fecal‒oral transmission. While Ypt causes acute gastroenteritis in humans, an association with Kawasaki disease (KD), a disease that primarily affects infants and young children and causes multisystemic vasculitis, has also been suspected. Although KD represents a significant health concern worldwide, the highest annual incidence rate is reported in Japan. Previously, a geographical origin-dependent population structure of Ypt comprising the Asian, transitional, and European clades was proposed. However, genomic data on KD-associated Ypt strains is currently unavailable. In this study, to analyze the phylogenetic and genomic features of KD-associated strains, we determined the whole-genome sequences of 35 Japanese Ypt strains, including 11 KD-associated strains, and constructed a genome set (n = 204) representing the global population of Ypt by adding publicly available Ypt genomes. In a phylogenetic analysis, all sequenced Japanese strains, including the KD-associated strains, belonged to the Asian clade, which appeared to be the ancestral clade of Ypt, and the KD-associated strains belonged to multiple lineages in this clade. Strains from patients with Far East scarlet-like fever (FESLF), a KD-related disease, also belonged to the Asian clade. Moreover, no KD strain-specific genes were identified in pan-genome-wide association study analyses. Notably, however, the gene encoding a superantigen called Yersinia pseudotuberculosis-derived mitogen (YPM) showed a distribution pattern highly biased to the Asian clade. Although further studies are needed, our results suggest that Asian clade strains may have a greater potential to trigger KD.

Paratyphoid B fever (PTB) is caused by an invasive lineage (phylogroup 1, PG1) of Salmonella enterica serotype Paratyphi B (SPB). However, little was known about the global population structure, geographic distribution, and evolution of this pathogen. Here, we report a whole-genome analysis of 568 historical and contemporary SPB PG1 isolates, obtained globally, between 1898 and 2021. We show that this pathogen existed in the 13th century, subsequently diversifying into 11 lineages and 38 genotypes with strong phylogeographic patterns. Following its discovery in 1896, it circulated across Europe until the 1970s, after which it was mostly reimported into Europe from South America, the Middle East, South Asia, and North Africa. Antimicrobial resistance recently emerged in various genotypes of SPB PG1, mostly through mutations of the quinolone-resistance-determining regions of gyrA and gyrB. This study provides an unprecedented insight into SPB PG1 and essential genomic tools for identifying and tracking this pathogen, thereby facilitating the global genomic surveillance of PTB.

Case 1: A 71-year-old man was admitted to our hospital with progressive fever and dyspnea, which had developed three days after the onset of COVID-19. Initial chest computed tomography (CT) revealed a pulmonary lesion consistent with a secondary bacterial infection. Streptococcus pyogenes was isolated from cultures of primary sputum collected from the endotracheal tube, and identified as the causative microorganism. Case 2: A 91-year-old man was transferred to our hospital with severe hypotension, which had developed nine days after the onset of COVID-19. A chest CT revealed pericardial effusion and pericardiocentesis yielded purulent fluid. S. pyogenes was isolated from the fluid specimens and was identified as the causative microorganism of the secondary bacterial pericarditis. Isolates from both patients were subsequently identified as M1UK-lineage S. pyogenes via genetic analysis. This report implicates COVID-19 as a potential risk factor for severe M1UK infection via the respiratory tract.

Escherichia albertii is an emerging enteropathogen and its distribution in various foods and environmental samples has been reported in many regions around the world. In this study, we aimed to identify effective isolation and detection methods for E. albertii in various foods and environmental water samples. E. albertii-specific polymerase chain reaction (PCR) was positive in chicken, oyster, river water, and wastewater samples, and E. albertii was isolated from these PCR-positive samples except the wastewater sample. E. albertii was not isolated from any of the samples without screening PCR; therefore, PCR is useful for the detection and isolation of E. albertii in foods and environmental water samples. The effect of two-step enrichment with four kinds of selective enrichment broth was compared with cycle threshold (Ct) values of the E. albertii-specific real-time PCR assay and the isolation results. The Ct values in three out of five samples were lower in the second enriched culture than those of the first enriched culture, and E. albertii was isolated from enriched cultures showed Ct values <25. These results suggest that the population of E. albertii in these three samples increased in the second enriched culture compared with the first enriched culture, and isolating E. albertii from an enriched culture showing Ct values <25 is an efficient method. Genetic analysis was performed to E. albertii isolates from food, environmental water, and human fecal samples, and all the isolates possessed eae, and isolates from chicken, pork, and river water samples showed the same EAOg type as E. albertii isolated from human fecal samples. Therefore, it was suggested that a continuous attention should be paid to E. albertii in food and environment.

Given the continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of new inhibitors is necessary to enhance clinical efficacy and increase the options for combination therapy for the coronavirus disease 2019. Because marine organisms have been a resource for the discovery of numerous bioactive molecules, we constructed an extract library of marine invertebrates collected from the Okinawa Islands. In this study, the extracts were used to identify antiviral molecules against SARS-CoV-2. Using a cytopathic effect (CPE) assay in VeroE6/TMPRSS2 cells, an extract from the marine sponge Theonella swinhoei was found to reduce virus-induced CPE. Eventually, onnamide A was identified as an antiviral compound in the extract using column chromatography and NMR analysis. Onnamide A inhibited several SARS-CoV-2 variant-induced CPEs in VeroE6/TMPRSS2 cells as well as virus production in the supernatant of infected cells. Moreover, this compound blocked the entry of SARS-CoV-2 pseudo-virions. Taken together, these results demonstrate that onnamide A suppresses SARS-CoV-2 infection, which may be partially related to entry inhibition, and is expected to be a candidate lead compound for the development of anti-SARS-CoV-2 drugs.

Abstract Shiga toxin (Stx)-producingEscherichia coli(STEC) causes serious gastrointestinal illness, including hemorrhagic colitis and hemolytic uremic syndrome. Although all known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages), the production of Stx2 is known to be a major risk factor for severe STEC infections. The production of Stx2, but not Stx1, is tightly coupled with the induction of Stx phages, and Stx2 production levels vary between STEC strains, even within the same serotype. Here, we analyzed the genomic diversity of all Stx phages in 71 strains representing the entire O145:H28 lineage, one of the major STECs. Our analysis revealed the highly dynamic nature of the Stx phages in O145:H28, including the independent acquisition of similar Stx phages by different sublineages and the frequent changes in Stx phages in the same sublineages due to the gain and loss of Stx phages. Analyses of Stx2 production levels in O145:H28 strains and K-12 lysogens of Stx2 phages of specific groups and types, which were defined by their early region sequences and CI repressors, respectively, revealed that short-tailed Stx2a phages (S-Stx2a phages) confer significantly greater Stx2 production to host strains than long-tailed Stx2a phages (L-Stx2a phages). However, L-Stx2a phages that encode a specific type of CI repressor promoted Stx2 production, comparable to the level of production among S-Stx2a phages, as well as promoted virulence to host strains, exceeding the level among other L-Stx2a phages. We also showed a clear link between the phage induction efficiency, which was primarily determined by the early region of each phage, and the level of Stx2 production by host strains. These results provide important insights into the diversification and dynamism of Stx phages and the relationship between the variations in Stx2 phages and the amount of Stx2 production by their host strains.Author summaryShiga toxin (Stx)-producingEscherichia coli(STEC) is an important human intestinal pathogen that causes severe illnesses. These bacteria produce Stx1, Stx2 or both toxins, but the production of Stx2 is an important measure of the virulence of STEC strains. While both types of Stx are encoded by bacteriophages (Stx phages), Stx2 production is tightly coupled with phage induction, and variations in Stx2 phages have been associated with variations in Stx2 production levels by their host O157:H7 STEC strains. However, in non-O157 STEC strains, the variation in Stx phages and its association with host strain production of Stx2 have not yet been fully analyzed. This systematic study of Stx phages in O145:H28 STEC reveals not only the marked genomic diversity and dynamism of Stx phages in this STEC lineage but also that short-tailed Stx2 phages and a specific group of long-tailed Stx2 phages induce high levels of Stx2 production by host strains, and this increased production is linked to the efficient induction of phages.