港 雄介

BACKGROUND: Flomoxef and cefmetazole have been reported to be effective against broad-spectrum β-lactamase-producing bacteria and have gained attention as a potential alternative to carbapenems. This study aimed to compare the efficacy of these two drugs in treating urinary tract infection (UTI) by integrating in vitro data and two real-world databases. METHODS: The susceptibility rates of third-generation cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae to flomoxef and cefmetazole were compared using comprehensive national antimicrobial resistance surveillance data. Combined antimicrobial activities against an extended-spectrum beta-lactamase (ESBL)-producing multidrug-resistant bacterial strain were tested by diagonal measurement of n-way drug interactions. The effectiveness of the two drugs in treating UTIs was compared using hospital stay duration data obtained from the Japan Medical Data Center (JMDC) Claims Database. RESULTS: Third-generation cephalosporin-resistant E. coli and K. pneumoniae, including ESBL-producing strains, were similarly susceptible to flomoxef and cefmetazole. In vitro assessment against an ESBL-producing multidrug-resistant strain revealed similar antimicrobial interaction patterns. JMDC Claims data analysis showed that the median hospital stay was 11 (95% confidence interval [CI]: 11-11) and 4 (95% CI: 3-5) days in the cefmetazole and flomoxef groups, respectively (log-rank test, P < 0.001). Moreover, the flomoxef group demonstrated a significantly lower frequency of adverse events such as C. difficile infection and renal failure. CONCLUSIONS: The effectiveness of flomoxef is comparable to that of cefmetazole in treating UTIs without major complications. Thus, flomoxef is a viable treatment option for UTIs in locales with a high prevalence of ESBL-producing strains.

OBJECTIVE: To examine the international trends for nontuberculous mycobacterial-associated mortality rates, as nontuberculous mycobacterial infections are becoming increasingly prevalent and pose a significant public health challenge, especially in older populations. METHODS: This retrospective observational study used data from the World Health Organization mortality database, which included patients with nontuberculous mycobacterial infection in 83 countries. We stratified the data by sex, age, and geographic region and calculated crude and age-standardized mortality rates to estimate long-term mortality trends. RESULTS: In total, 42,182 nontuberculous mycobacterial infection-associated deaths (58.1% in women) were reported in 83 countries between 2000 and 2022. The locally weighted regression model estimation for the nontuberculous mycobacterial infection-associated mortality rate more than doubled-from 0.36 deaths per 1,000,000 individuals in 2000 to 0.77 deaths per 1,000,000 individuals in 2022. Eighty-six percent of nontuberculous mycobacterial infection-associated deaths occurred in people aged ≥65 years. The mortality rate was the highest in the Western Pacific Region. CONCLUSIONS: This study highlights the impact of emerging nontuberculous mycobacterial diseases and the importance of targeted interventions for managing and reducing mortality, particularly in vulnerable older populations. Further studies are warranted to determine the factors contributing to geographical disparity and treatment options.

This study highlights the novel potential of molecular aggregates as inhibitors of a disease-related protein. Enzyme inhibitors have been studied and developed as molecularly targeted drugs and have been applied for cancer, autoimmune diseases, and infections. In many cases, enzyme inhibitors that are used for therapeutic applications interact directly with enzymes in a molecule-to-molecule manner. We found that the aggregates of a small compound, Mn007, inhibited bovine pancreatic DNase I. Once Mn007 molecules formed aggregates, they exhibited inhibitory effects specific to DNases that require divalent metal ions. A DNase secreted by Streptococcus pyogenes causes streptococcal toxic shock syndrome (STSS). STSS is a severe infectious disease with a fatality rate exceeding 30% in patients, even in this century. S. pyogenes disrupts the human barrier system against microbial infections through the secreted DNase. Until now, the discovery/development of a DNase inhibitor has been challenging. Mn007 aggregates were found to inhibit the DNase secreted by S. pyogenes, which led to the successful suppression of S. pyogenes growth in human whole blood. To date, molecular aggregation has been outside the scope of drug discovery. The present study suggests that molecular aggregation is a vast area to be explored for drug discovery and development because aggregates of small-molecule compounds can inhibit disease-related enzymes.

The development of new antibacterial drugs with different mechanisms of action is urgently needed to address antimicrobial resistance. MraY is an essential membrane enzyme required for bacterial cell wall synthesis. Sphaerimicins are naturally occurring macrocyclic nucleoside inhibitors of MraY and are considered a promising target in antibacterial discovery. However, developing sphaerimicins as antibacterials has been challenging due to their complex macrocyclic structures. In this study, we construct their characteristic macrocyclic skeleton via two key reactions. Having then determined the structure of a sphaerimicin analogue bound to MraY, we use a structure-guided approach to design simplified sphaerimicin analogues. These analogues retain potency against MraY and exhibit potent antibacterial activity against Gram-positive bacteria, including clinically isolated drug resistant strains of S. aureus and E. faecium. Our study combines synthetic chemistry, structural biology, and microbiology to provide a platform for the development of MraY inhibitors as antibacterials against drug-resistant bacteria.

The total synthesis of capuramycin (1), which is a promising anti-tubercular antibiotics, has been accomplished using Ferrier-type I reaction as a key step. This total synthesis is an alternative approach to the synthesis of capuramycin and its analogues. The 3'-O-demethyl analogue (2), which exhibits an equivalent antibacterial activity as capuramycin (1) against Mycobacterium smegmatis and Mycobacterium avium, is suggested to have potential as a lead structure of capuramycin analogues because 2 is more accessible from a synthetic view point.

Tuberculosis (TB) is a leading source of infectious disease mortality globally. Antibiotic-resistant strains comprise an estimated 10% of new TB cases and present an urgent need for novel therapeutics. β-lactam antibiotics have traditionally been ineffective against M. tuberculosis (Mtb), the causative agent of TB, due to the organism's inherent expression of β-lactamases that destroy the electrophilic β-lactam warhead. We have developed novel β-lactam conjugates, which exploit this inherent β-lactamase activity to achieve selective release of pyrazinoic acid (POA), the active form of a first-line TB drug. These conjugates are selectively active against M. tuberculosis and related mycobacteria, and activity is retained or even potentiated in multiple resistant strains and models. Preliminary mechanistic investigations suggest that both the POA "warhead" as well as the β-lactam "promoiety" contribute to the observed activity, demonstrating a codrug strategy with important implications for future TB therapy.

It is important to understand and control the biologically active conformation in medicinal chemistry. Muraymycins and caprazamycins, which are strong inhibitors of MraY, are promising antibacterial agents with a novel mode of action. Focusing on a sugar puckering and a dihedral angle ϕ of the uridine moiety of these natural products, LNA/BNA-type 5'-O-aminoribosyluridine analogues, whose puckering of the ribose moiety are completely restricted to the N-type, were designed and synthesized as simplified MraY inhibitors. Their conformation-activity relationship was further investigated in details. The conformation-activity relationship analysis investigated in this study could be a general guideline for simplification and rational drug design of MraY inhibitory nucleoside natural products.

Pyrazinamide (PZA) plays a crucial role in first-line tuberculosis drug therapy. Unlike other antimicrobial agents, PZA is active against Mycobacterium tuberculosis only at low pH. The basis for this conditional drug susceptibility remains undefined. In this study, we utilized a genome-wide approach to interrogate potentiation of PZA action. We found that mutations in numerous genes involved in central metabolism as well as cell envelope maintenance and stress response are associated with PZA resistance. Further, we demonstrate that constitutive activation of the cell envelope stress response can drive PZA susceptibility independent of environmental pH. Consequently, exposure to peptidoglycan synthesis inhibitors, such as beta-lactams and d-cycloserine, potentiate PZA action through triggering this response. These findings illuminate a regulatory mechanism for conditional PZA susceptibility and reveal new avenues for enhancing potency of this important drug through targeting activation of the cell envelope stress response. IMPORTANCE For decades, pyrazinamide has served as a cornerstone of tuberculosis therapy. Unlike any other antitubercular drug, pyrazinamide requires an acidic environment to exert its action. Despite its importance, the driver of this conditional susceptibility has remained unknown. In this study, a genome-wide approach revealed that pyrazinamide action is governed by the cell envelope stress response. This observation was validated by orthologous approaches that demonstrate that a central player of this response, SigE, is both necessary and sufficient for potentiation of pyrazinamide action. Moreover, constitutive activation of this response through deletion of the anti-sigma factor gene rseA or exposure of bacilli to drugs that target the cell wall was found to potently drive pyrazinamide susceptibility independent of environmental pH. These findings force a paradigm shift in our understanding of pyrazinamide action and open new avenues for improving diagnostic and therapeutic tools for tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

BACKGROUND AND AIM: The current prevalence of hepatitis C virus infection and hepatitis C virus-associated mortality in Japan falls short of the World Health Organization goal of viral hepatitis elimination by 2030. We aimed to evaluate the trends in hepatitis C virus-associated mortality in Japan. METHODS: This nationwide observational study used the Japanese Vital Statistics from 1998 to 2017 and included all Japanese hepatitis C virus-associated deaths (84 936) of adults aged ≥ 40 years. We calculated the crude and age-standardized mortality rates per 100 000 persons by age and sex. Joinpoint regression analysis was used to identify significant changing points in trends and to estimate the annual percentage changes and the average annual percentage changes for the entire study period. RESULTS: The crude mortality rate per 100 000 persons (annual death number) increased from 5.5 (3548) in 1998 to 7.0 (4843) in 2005 and decreased to 4.0 (3095) in 2017. By 2017, the crude mortality rates per 100 000 persons among men and women had dropped to 3.6 and 4.3, respectively. The age-standardized mortality rate was higher in women than in men. The average annual percentage change was -3.8% (95% confidence interval: -5.0 to -2.5). The declining trend was more rapid in men (-4.5%, 95% confidence interval: -5.3 to -3.6) than in women (-2.7%, 95% confidence interval: -3.8 to -1.6). CONCLUSIONS: Trends in hepatitis C virus-associated mortality rates have declined in an accelerating manner in Japan, especially among men.

<title>Abstract</title>The global incidence of the human nontuberculous mycobacteria (NTM) disease is rapidly increasing. However, knowledge of gene essentiality under optimal growth conditions and conditions relevant to the natural ecology of NTM, such as hypoxia, is lacking. In this study, we utilized transposon sequencing to comprehensively identify genes essential for growth in <italic>Mycobacterium intracellulare</italic>. Of 5126 genes of <italic>M. intracellulare</italic> ATCC13950, 506 genes were identified as essential genes, of which 280 and 158 genes were shared with essential genes of <italic>M. tuberculosis</italic> and <italic>M. marinum</italic>, respectively. The shared genes included target genes of existing antituberculous drugs including SQ109, which targets the trehalose monomycolate transporter MmpL3. From 175 genes showing decreased fitness as conditionally essential under hypoxia, preferential carbohydrate metabolism including gluconeogenesis, glyoxylate cycle and succinate production was suggested under hypoxia. Virulence-associated genes including proteasome system and mycothiol redox system were also identified as conditionally essential under hypoxia, which was further supported by the higher effective suppression of bacterial growth under hypoxia compared to aerobic conditions in the presence of these inhibitors. This study has comprehensively identified functions essential for growth of <italic>M. intracellulare</italic> under conditions relevant to the host environment. These findings provide critical functional genomic information for drug discovery.

&lt;p&gt;Metabolomics is an “omics” approach to quantitatively measure a large set of metabolites. In this chapter, we describe an example method for performing liquid chromatography coupled to mass spectrometry (LC–MS)-based untargeted metabolomics on a cell extract from Vibrio cholerae.&lt;/p&gt;