Yasufumi Matsumura

Chryseobacterium species are environmental bacteria occasionally implicated in human infections; however, their clinical and genomic features are not fully characterized. This study investigated the clinical, genotypic, and phenotypic characteristics of Chryseobacterium strains collected through the MultiDrug-Resistant Organisms Clinical Research Network, a multicenter prospective study of patients with carbapenem-resistant gram-negative bacilli isolated at tertiary care hospitals in Japan between April 2019 and March 2024. Among 18 patients with Chryseobacterium spp. confirmed by whole-genome sequencing (WGS), 5 had infection, and 13 were colonized. Bacteremia and pneumonia were the common types of infection. The isolates comprised five species, with Chryseobacterium indologenes and Chryseobacterium culicis being the most frequently detected. Compared with WGS, 16S rRNA gene sequencing demonstrated the highest species-level identification accuracy (93.8%), followed by MALDI Biotyper (50.0%), VITEK-MS (31.3%), and VITEK 2 Blue (31.3%). All systems correctly identified C. indologenes , but other species were frequently misidentified or remained unidentified because of limited database coverage. All isolates exhibited broad β-lactam resistance, including resistance to carbapenems, which was associated with species-specific combinations of metallo-β-lactamase and extended-spectrum β-lactamase genes. Minocycline, trimethoprim-sulfamethoxazole, and cefiderocol retained activity across all species. These findings highlight that diverse Chryseobacterium species, including uncommon ones, can be associated with human infections. Incorporating genomic approaches such as WGS or 16S rRNA gene analysis can improve species-level identification, especially for rare or emerging species. IMPORTANCE: Chryseobacterium species are emerging opportunistic pathogens that pose a diagnostic and therapeutic challenge as they are intrinsically resistant to most β-lactams, including carbapenems, and are often misidentified by routine clinical laboratory systems. In this multicenter study, we show that clinical Chryseobacterium isolates represent greater species diversity than is typically recognized, extending beyond Chryseobacterium indologenes to include uncommon species associated with both infection and colonization. Whole-genome sequencing clarified species assignments, revealed species-specific β-lactamase profiles, and highlighted the limitations of commonly used identification platforms, particularly for species absent from commercial databases. Despite broad β-lactam resistance, cefiderocol, minocycline, and trimethoprim-sulfamethoxazole retained in vitro activity across species. These findings emphasize the need for genomic or sequence-based identification to accurately define the clinical spectrum of Chryseobacterium species, guide interpretation of antimicrobial susceptibility, and improve recognition of these organisms as emerging healthcare-associated pathogens.

During the 2010s, Escherichia coli ST131 clade A tested sensitive to most antibiotics. In the 2020s, Europe experienced outbreaks of fluoroquinolone-resistant ST131-A that contained CTX-M β-lactamases and OXA-48-like carbapenemases. We investigated the acquisition of different antimicrobial resistance determinants in the emergence of multidrug-resistant (MDR) ST131-A. Phylogenetic analysis and Bayesian inference were utilized to define subclades and clusters (within subclades) and investigate the evolutionary history of ST131-A, using sequenced genomes and publicly available genomic data ( n =2,905). ST131-A consisted of five dominant subclades and ten frequent clusters, representing 93 and 83% of the total population, respectively. The sampled most recent common ancestors of the dominant subclades dated mainly to the 1990s. Several MDR clusters linked with diverse quinolone resistance-determining region mutations, CTX-M and OXA-48-like genes diversified during the 2000s. The distribution and mobile genetic elements linked with bla _CTX-M-14 , bla _CTX-M-15 and bla _CTX-M-27 differed among ST131-A subclades and clusters across geographic locations. Three ST131-A clusters belonging to different subclades and carrying bla _OXA-48 and bla _OXA-244 in distinct genetic contexts were associated with the recent European dissemination. The identification of MDR ST131-A subclades and clusters with divergent molecular architectures emerging independently across regions highlights the challenges for public health surveillance and control efforts.

Escherichia coli with penicillin-binding protein 3 (PBP3) insertions appeared in the early 2000s and expanded during the 2010s. The YRIN and YRIK variants predominate globally, while other insertion types remain rare. These insertions frequently co-occur with carbapenemases (NDM-5 and OXA-181) and high-risk clones (ST410, ST167, and ST405), contributing to the evolution and dissemination of multidrug resistance. Newer β-lactam/β-lactamase inhibitor combinations (ceftazidime/avibactam, aztreonam/avibactam, and cefepime/taniborbactam) and cefiderocol with affinities for PBP3 show reduced activity against E. coli with YRIN and YRIK insertions. The continued spread of such strains threatens the effectiveness of these β-lactam agents. In contrast, agents targeting PBP2, such as cefepime/zidebactam and cefiderocol-xeruborbactam, retain potent in vitro activity and will likely be important for future treatment strategies. Laboratory methods to detect PBP3 insertions are lacking, raising concern that these strains may be spreading largely undetected. Current surveillance efforts are insufficient, leaving significant gaps in data describing the global distribution and prevalence of E. coli with PBP3 insertions. The convergence of β-lactamase production and PBP target modification represents a paradigm shift within E. coli toward multidrug resistance. This occurrence provides a major barrier to current and future β-lactam/β-lactamase inhibitor development. There is an urgent need for enhanced global surveillance, along with rapid diagnostic tools capable of identifying PBP3 insertions and associated high-risk clones among carbapenemase-producing E. coli . Future strategies should prioritize agents with activities against multiple essential PBPs to preserve β-lactam efficacy.

Purpose: Although indications for anti-interleukin 6 (IL-6) receptor antibody (anti-IL6R) therapy continue to expand, the clinical features of bloodstream infection (BSI) in anti-IL6R recipients remain poorly characterized. We investigated the prevalence, clinical and microbiological features of BSI among the anti-IL6R recipients. Methods: We conducted a multicenter, retrospective study of BSI episodes occurring within 90 days of anti-IL6R administration between 2005 and 2024. Complicated BSI was defined as the presence of either (1) infection-related mortality, (2) remote or localized suppurative complications, (3) embolic stroke, or (4) recurrence within 90 days. Associations between clinical variables and complicated BSI were analyzed. Results: Among 922 anti-IL6R recipients, 47 patients (5%) developed BSI. Demographics varied by indication for anti-IL6R, and 89% received concomitant immunosuppressive therapy. Catheter-related bloodstream (23%) and intra-abdominal (21%) were most common foci, followed by bone and joint (14%). At presentation, fever was absent in 53%, and serum CRP was within normal range in 30%. Septic shock occurred in 26%, and all-cause 30-day mortality was 19%. Complicated BSI occurred in 22 episodes (47%). In univariate analysis, underlying rheumatic disease, community acquisition, onset within 21 days of anti-IL6R administration, and Staphylococcus aureus or Klebsiella pneumoniae etiology were associated with complicated BSI, whereas higher pulse rate at presentation and Pitt bacteremia score ≥ 4 were inversely associated. Conclusions: In anti-IL6R recipients, BSI frequently lacked typical signs such as fever but often resulted in severe outcomes. Clinicians should maintain a high index of suspicion for complicated BSI in this population.

We aimed to identify genomically defined strain transmission events of carbapenem-resistant Gram-negative bacteria (CRGNB) in hospitals, estimate species-specific transmission rates and identify risk factors for transmission. The study included 708 patients from a Japanese prospective multicentre cohort study, the MultiDrug-Resistant organisms clinical research network, from whom at least one CRGNB isolate was collected between 2019 and 2024 (750 isolates in total). Clinical data were retrieved from electronic medical records, and microbiological analyses were performed at a central reference laboratory. Transmission events were determined via whole-genome sequencing data and single-nucleotide polymorphism-based genomic clustering. We identified 37 genomic clusters associated with intrahospital transmission and 1 associated with interhospital transmission. Ninety-four patients (13%) carried at least one isolate involved in intrahospital transmission, and each transmission event involved two to six patients. Patients with Acinetobacter spp. (60% involved in intrahospital transmission), Enterobacter cloacae complex (23%) and Klebsiella oxytoca complex (59%) were significantly associated with intrahospital transmission and were considered high-risk species groups. Patients with Enterobacterales isolates carrying acquired carbapenemase genes had higher intrahospital transmission rates relative to those without (31% vs. 2%). In multivariable analysis, independent risk factors for patients with intrahospital transmission were overlap in ward and time with other CRGNB-positive patients, carriage of high-risk species groups and acquired carbapenemase genes. In this Japanese multicentre cohort, intrahospital transmission of CRGNB was associated with both microbiological characteristics and patient- and healthcare-related factors, underscoring the need for targeted surveillance and infection prevention strategies.

Respiratory viruses, such as influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remain major global health threats, and the emergence of drug-resistant variants underscores the urgent need for host-targeted antiviral strategies. Prohibitins (PHBs), mitochondrial scaffold proteins involved in diverse cellular processes, are host factors exploited by multiple viruses. Here, we investigated the antiviral potential of two PHB-binding triazine melanogenin derivatives, Mel56 and Mel6, against IAV and SARS-CoV-2. Mel56, but not Mel6, exhibited potent anti-IAV activity. In particular, Mel56 increased cell survival, suppressed viral nucleoprotein expression, and reduced viral gene transcription in IAV-infected Madin-Darby canine kidney (MDCK) cells and A549 human lung carcinoma cells. PHB2 knockdown enhanced the inhibitory effect of Mel56, supporting the involvement of PHBs in its antiviral mechanism. Transcriptomic profiling revealed that Mel56 downregulates virus-induced immune response genes while upregulating antioxidant response-related genes, including nuclear factor erythroid 2-related factor 2 (NRF2) target genes. NRF2 activation by Mel56 was confirmed using a reporter assay. Consistent with these findings, Mel56 impaired mitochondrial ATP synthesis and electron transport in isolated rat liver mitochondria and attenuated mitochondrial membrane potential and promoted mitochondrial reactive oxygen species production in live MDCK cells. Importantly, Mel56 exhibited potent anti-SARS-CoV-2 activity in human induced pluripotent stem cell-derived lung organoids. Mel56 did not upregulate NRF2 target genes in SARS-CoV-2-infected organoids. These findings identify Mel56 as a PHB-binding compound with broad-spectrum antiviral activity and support the development of PHB-targeting ligands as a novel host-directed therapeutic strategy against respiratory viral infections. IMPORTANCE: This study identifies Mel56 as a novel host-directed antiviral compound that targets prohibitins and suppresses both influenza A virus and severe acute respiratory syndrome coronavirus 2. Mel56 exhibits broad-spectrum antiviral activity in conventional cell culture models as well as in physiologically relevant human lung organoids. Mechanistically, Mel56 upregulates antioxidant response-related genes, activates nuclear factor erythroid 2-related factor 2, impairs mitochondrial function, and enhances mitochondrial reactive oxygen species production. Notably, its effects differ depending on the cellular context, underscoring the complexity of host signaling pathways during viral infection. These findings highlight prohibitins as promising therapeutic targets and provide proof of concept for the development of host-directed strategies to combat respiratory viruses and mitigate the emergence of drug resistance.

Compared with microscopy, quantitative real-time PCR (qPCR) is more sensitive for the diagnosis of Pneumocystis jirovecii pneumonia (PcP) and may also help distinguish PcP from colonization based on fungal burden. In this retrospective cohort study, we evaluated the performance of the LeaDEA Pneumocystis PCR Kit, a single-packaged reagent system designed for use on a fully automated molecular testing platform, using respiratory specimens from immunocompromised patients. The analytical and clinical performance of the LeaDEA assay on the GeneLEAD VIII platform was compared with that of a previously described laboratory-developed reference qPCR assay targeting the major surface glycoprotein gene of P. jirovecii. The limit of detection was 500 copies/ml for both assays, with a quantification range of 10³–10⁸ copies/ml. Among 350 patients (158 bronchoalveolar lavage and 192 sputum [including induced sputum] samples), the reference assay-based European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) criteria classified 162 as having PcP (13 proven and 149 probable) and 188 as non-PcP. The overall concordance of qPCR results between the assays was 93% (kappa, 0.85). Compared with the reference assay-based diagnoses, the LeaDEA assay-based diagnoses showed 92.0% positive % agreement and 96.3% negative % agreement (kappa, 0.88). Among the PCR-positive non-HIV patients, the copy numbers measured by the LeaDEA assay distinguished PcP from colonization based on qPCR-independent diagnoses, with 63.8% sensitivity and 72.5% specificity, at an optimal cutoff of 20 427 copies/ml. The LeaDEA assay demonstrated analytical and clinical performance comparable to that of a reference qPCR assay, which may facilitate the diagnosis of PcP in routine clinical laboratories.

Carbapenem-resistant Enterobacterales (CRE) pose a global threat due to limited treatment options and high mortality. Difficult-to-treat resistance (DTR), defined as non-susceptibility to all conventional β-lactams and fluoroquinolones, has primarily been applied to Pseudomonas aeruginosa . However, its relevance for Enterobacterales remains unclear, particularly in regions with a distinct carbapenemase landscape, such as Japan where IMP-type metallo-β-lactamases predominate. We analyzed the MultiDrug-Resistant organisms clinical research network (MDRnet) cohort, a multicenter prospective study conducted at 13 Japanese hospitals between April 2019 and March 2024. This analysis included patients with clinically indicated cultures yielding CRE. Clinical characteristics and outcomes assessed using the desirability of outcome ranking (DOOR) framework and genomic epidemiology characterized by whole-genome sequencing were compared between DTR and non-DTR groups. Among 196 CRE cases, 64 (32.7%) represented infections, including 12 DTR cases (18.8%). Carbapenemase genes were detected in 34/64 infections (53.1%), with similar prevalence in the DTR and non-DTR groups (50.0% vs 53.8%). bla _IMP-1 was the most frequently identified carbapenemase gene ( n = 28). The overall 30-day mortality rate was 21.9%, with 16.7% (95% confidence interval [CI], 2.1%–48.4%) in the DTR group and 23.1% (95% CI, 12.5%–36.8%) in the non-DTR group. DOOR outcomes were similar between groups. Appropriate empiric and definitive therapy was less frequently administered in the DTR group. In this cohort, where IMP-type carbapenemases and non-DTR CRE are prevalent, DTR classification did not appear to correlate with 30-day mortality or DOOR outcomes. These findings underscore the importance of regional molecular epidemiology when interpreting clinical outcomes of CRE infections. IMPORTANCE: Difficult-to-treat resistance (DTR) is increasingly used to assess the clinical impact of antimicrobial resistance, but its significance in carbapenem-resistant Enterobacterales (CRE) may vary across molecular epidemiologic settings. In this prospective multicenter study from Japan, where IMP-type carbapenemases predominated, most CRE infections were classified as non-DTR. Whole-genome sequencing also revealed a distinct local clonal structure. DTR was not associated with 30-day mortality or desirability of outcome ranking despite lower rates of appropriate empiric and definitive therapy. Our study highlights the importance of interpreting DTR in CRE within the context of regional molecular epidemiology and supports the need for continued regional multicenter studies to evaluate its clinical utility.