Classification of the metallo ß-lactamase subtype produced by the carbapenem-resistant Pseudomonas aeruginosa isolates in Japan.

INTRODUCTION: Multidrug resistant microorganisms are a serious threat to human health. Under the circumstances, a front line of antimicrobials in clinical setting may be carbapenem ß-lactams (CRBP). However, emergence of CRBP resistant (CRBP-r) Gram-negative bacteria are the most alarming. CRBP-r is mainly caused to the production of ß-lactamase, down and up expression of the diffusion channel and the efflux pump genes, respectively. Among them, production of metallo-ß-lactamase (MBL) is a major cause of high-level of CRBP-r. METHOD: We analyzed the MBL subtypes by PCR and DNA sequencing in CRBP-r Psudomonas aeruginosa in the collection of the joint program by the Japanese Association for Infectious Diseases, Japan Society for Clinical Microbiology and Japanese Society of Chemotherapy (2006-2015 in Japan). RESULTS: Among 275 strains out of a total 1716 isolates, 23 (8.3%) were MBL-positive exhibiting resistant to meropenem (MEPM), imipenem, ceftazidime, cefepime, ciprofloxacin and levofloxacin without exception and the MIC of MEPM appeared over 128 µg/mL. Their MBL subtype analysis revealed that 16, 2, and 2 isolates were IMP-1, IMP-7 and VIM-2 positive, respectively, and one isolate each expressed either IMP-10, IMP-34 or IMP-41. CONCLUSIONS: This study revealed that all the MBL-positive CRBP-r isolates were highly resistant to carbapenems dominating IMP-1 production.

[NEED FOR SUBCUTANEOUS IMMUNOTHERAPY IN PEDIATRIC PATIENTS WITH ALLERGIC RHINITIS AFTER THE EXPANDED INDICATION FOR SUBLINGUAL IMMUNOTHERAPY].

BACKGROUND: Although the indication for sublingual immunotherapy (SLIT) was expanded in pediatric patients with allergic rhinitis in recent years, some patients choose subcutaneous immunotherapy (SCIT). OBJECTIVE: The objective of this study was to investigate the reason why they chose SCIT in spite of injection pain and high risk of systemic reaction. METHODS: The subjects were 58 patients who diagnosed with allergic rhinitis and introduced SCIT at our hospital between June 2018 and January 2021. The reasons why they chose SCIT were collected from medical records. RESULT: The median age at the start of treatment was 9 years 0 months. The most common reason why they chose SCIT was "difficult to maintain adherence of SLIT" in 28 cases (48%). Other reasons were as follows: "side effects of SLIT", "expectation for the effect of SCIT", "young age (under 5 years old)" and "motivation for allergic disease treatment". CONCLUSION: Two-thirds of the reasons why they chose SCIT were because it was difficult to continue SLIT for some reasons.

Real-time imaging of histone H4 hyperacetylation in living cells.

To visualize histone acetylation in living cells, we developed a genetically encoded fluorescent resonance energy transfer (FRET)-based indicator. Response of the indicator reflects changes in the acetylation state of both K5 and K8 in histone H4. Using this acetylation indicator, we were able to monitor the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors.

Conventional culture methods with commercially available media unveil the presence of novel culturable bacteria.

Recent metagenomic analysis has revealed that our gut microbiota plays an important role in not only the maintenance of our health but also various diseases such as obesity, diabetes, inflammatory bowel disease, and allergy. However, most intestinal bacteria are considered 'unculturable' bacteria, and their functions remain unknown. Although culture-independent genomic approaches have enabled us to gain insight into their potential roles, culture-based approaches are still required to understand their characteristic features and phenotypes. To date, various culturing methods have been attempted to obtain these 'unculturable' bacteria, but most such methods require advanced techniques. Here, we have tried to isolate possible unculturable bacteria from a healthy Japanese individual by using commercially available media. A 16S rRNA (ribosomal RNA) gene metagenomic analysis revealed that each culture medium showed bacterial growth depending on its selective features and a possibility of the presence of novel bacterial species. Whole genome sequencing of these candidate strains suggested the isolation of 8 novel bacterial species classified in the Actinobacteria and Firmicutes phyla. Our approach indicates that a number of intestinal bacteria hitherto considered unculturable are potentially culturable and can be cultured on commercially available media. We have obtained novel gut bacteria from a healthy Japanese individual using a combination of comprehensive genomics and conventional culturing methods. We would expect that the discovery of such novel bacteria could illuminate pivotal roles for the gut microbiota in association with human health.

Rapid and affordable size-selected PacBio single-molecule real-time sequencing template library construction using the bead-beating DNA extraction method.

This study demonstrated that bead-beating method facilitates a simple and rapid protocol for genomic DNA isolation for Pacific BioSciences (PacBio) sequencing with library construction of sufficient length. The protocol may also be beneficial for inactivating pathogens by simultaneous and instant DNA fragmentation, with no special equipment required to obtain large DNA fragments. This protocol was comparable in terms of quality to the standard protocol suggested by PacBio and represents an alternative, rapid shortcut for performing accurate PacBio sequencing.

Real-time imaging of histone H4K12-specific acetylation determines the modes of action of histone deacetylase and bromodomain inhibitors.

Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.