鈴木 匡弘

ABSTRACT With the widespread use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the number of identifiable bacterial species has increased. However, anaerobic bacteremia remains challenging to accurately diagnose due to the diverse range of anaerobic bacteria and the frequent occurrence of polymicrobial infections. Consequently, MALDI-TOF MS often fails to achieve accurate species-level identification in such cases. To address this limitation, we evaluated whole-genome sequencing (WGS) as an alternative method for identifying anaerobic bacteria in blood cultures. Over a 4-year period (April 2020 to March 2024), 69 cases of anaerobic bacteremia were identified, involving 85 bacterial strains. WGS successfully identified 73 strains (89%) at the species level. MALDI-TOF MS accurately identified 43 strains (59%) at the species level and 6 strains (8.2%) at the genus level. Among the 24 discordant strains, 9 species were not included in the database, and 6 species had limited prior reports of bloodstream infections. Additionally, 21 of the 69 cases (30%) were polymicrobial, and WGS revealed 9 cases (13%) in which multiple species had not been identified by MALDI-TOF MS. These results highlight the limitations of MALDI-TOF MS in anaerobic bacterial identification, particularly in polymicrobial infections, and suggest that alternative molecular approaches are necessary to improve diagnostic accuracy. IMPORTANCE Accurate identification of anaerobic bacteria remains a significant challenge despite the widespread use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. While this technology has improved the detection of many bacterial species, some anaerobes remain unidentified due to their absence from reference databases and the difficulties associated with their isolation, particularly in polymicrobial infections. Whole-genome sequencing (WGS) has revealed previously unreported anaerobes and identified polymicrobial infections that were initially misclassified as monomicrobial. Our findings underscore the importance of implementing molecular approaches such as WGS- or PCR-based methods in clinical diagnostics to improve the detection of anaerobic pathogens.

The genus Aeromonas is increasingly implicated in human infections. However, accurate species-level identification remains challenging, particularly in clinical microbiology laboratories. This study aimed to develop a multiplex polymerase chain reaction (PCR) assay to identify four Aeromonas species-Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii, and Aeromonas dhakensis-most frequently associated with human infectious diseases. A total of 788 whole genome sequencing (WGS) data sets from 31 Aeromonas species were analyzed to identify open reading frames (ORFs) specifically present in A. hydrophila, A. caviae, A. veronii, and A. dhakensis. Primer sets were designed based on sequences of ORFs specific to each species to develop a multiplex PCR assay. To validate the efficacy of the assay, 256 clinical Aeromonas isolates were tested, and the results were compared with taxonomic affiliation inferred by WGS data, along with 19 type strains. The multiplex PCR successfully identified all strains of the four target species and produced no amplification in non-target species strains except the band for internal control. The multiplex PCR enables rapid and reliable identification of four Aeromonas spp. commonly involved in human infectious diseases.IMPORTANCEThe multiplex PCR assay facilitates accurate identification of clinically important Aeromonas spp. in clinical microbiology laboratories, providing crucial information to guide appropriate antimicrobial therapy and advance understanding of the epidemiology of Aeromonas spp.

BACKGROUND AND OBJECTIVES: Carbapenem-resistant Gram-negative bacilli (CRGNB), especially Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii, are critical pathogens associated with excess morbidity and mortality. To elucidate their molecular epidemiology and clinical outcomes in Japan, patients with CRGNB were enrolled in the MDR organisms clinical research network (MDRnet) consisting of eight tertiary care facilities. METHODS: Between 2019 and 2022, 246 unique patients with carbapenem-resistant Enterobacterales (CRE), carbapenem-resistant P. aeruginosa (CRPA) and carbapenem-resistant A. baumannii (CRAB) isolates were prospectively enrolled. RESULTS: A total of 246 isolates were collected from 246 patients, including 78 (31.7%) CRE, 167 (67.9%) CRPA and 1 (0.4%) CRAB. For CRE, 74.4% of the isolates carried carbapenemase genes with predominance of bla IMP (64.1%). Only 2.4% of CRPA had carbapenemase genes, which was lower than CRE. Among the infected patients, 20.0% and 12.5% died of CRE and CRPA within 30 days, respectively. In patients with CRE, the mortality rate within 30 days for those without carbapenemase-producing Enterobacterales (CPE) was higher compared with those with CPE (22.2% compared with 18.8%). CONCLUSIONS: Our study highlights the unique molecular epidemiology and clinical outcomes of CRGNB in Japan.

Abstract Patients with hematologic diseases have experienced COVID-19 with prolonged, progressive course. Here we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high SARS-CoV-2 levels persisted for more than 4 weeks, and two of them succumbed to COVID-19. Autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping CMV, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent acquisition of drug resistance and improve outcome, such as selection of appropriate treatment strategies for lymphoma considering patients’ immune status and institution of early intensive antiviral therapy.

OBJECTIVES: Infections caused by carbapenemase-producing Gram-negative pathogens have become a significant global public health challenge due to limited treatment options. Pathogens producing metallo-β-lactamase are particularly problematic since they are not inhibited by conventional β-lactamase inhibitors. Herein, we assess the in vitro activity of aztreonam in combination with relebactam against a collection carbapenemase producing organisms, including strains producing both serine-β-lactamase and IMP-type metallo-β-lactamase that are commonly encountered in Japan. METHODS: A total of 119 carbapenemase-producing clinical isolates were used in this study. Minimum inhibitory concentrations (MICs) of aztreonam and imipenem alone and aztreonam/relebactam, aztreonam/avibactam and imipenem/relebactam combinations were determined by the broth microdilution method. RESULTS: Aztreonam MICs were reduced in combination with relebactam for strains producing ESBL or AmpC in addition to IMP-type, NDM-type, GES-type or OXA-48 carbapenemases and for Stenotrophomonas spp. Additionally, aztreonam/relebactam combination MICs were significantly lower than MICs of aztreonam alone among IMP producers, NDM producers and Stenotrophomonas spp. Significant differences between aztreonam/relebactam and aztreonam MICs were also observed for strains of E. coli, K. pneumoniae and Enterobacter spp., many of which produced both metallo-β-lactamase and serine-β-lactamase. The aztreonam/relebactam combination showed comparable to higher MICs compared with the aztreonam/avibactam combination. CONCLUSION: The addition of relebactam has a potential to restore the activity of aztreonam against strains that produce metallo-β-lactamase and serine-β-lactamase. The combination may have a role in the treatment of infections due to these strains in countries without access to ceftazidime-avibactam.