Masahiro Suzuki

INTRODUCTION: Carbapenem-resistant Gram-negative bacteria (CRGNB) pose a major clinical threat. This study evaluated the in vitro activity of cefiderocol and other recently approved β-lactam/β-lactamase inhibitor combinations against major CRGNB. MATERIALS AND METHODS: A total of 292 CRGNB clinical isolates were analyzed, comprising 146 Enterobacterales, 106 Pseudomonas aeruginosa, and 40 Stenotrophomonas maltophilia, all collected from hospitals across Japan. Antimicrobial susceptibility testing was performed by broth microdilution (BMD). Disk diffusion testing was also conducted for cefiderocol, and categorical agreement with BMD was assessed. Whole-genome sequencing (WGS) was used for species confirmation and characterization of resistance determinants. RESULTS: Carbapenemase producers accounted for 64.4 % of Enterobacterales (94/146) and 8.5 % of P. aeruginosa (9/106), with metallo-β-lactamase (MBL) producers comprising 92.6 % (87/94) and 77.8 % (7/9), respectively. Based on CLSI breakpoints, 94.5 % (276/292) of isolates were susceptible to cefiderocol, including 91.8 % of Enterobacterales, 99.1 % of P. aeruginosa, and 92.5 % of S. maltophilia. Ceftolozane-tazobactam, ceftazidime-avibactam, and imipenem-relebactam were active against 12.3 %, 44.5 % and 45.9 % of Enterobacterales, and 89.6 %, 86.8 % and 72.6 % of P. aeruginosa, respectively. Categorical agreement between cefiderocol disk diffusion and BMD exceeded 92 % across all groups, although very major errors occurred in Enterobacterales (n = 2) and S. maltophilia (n = 3). Cefiderocol-non-susceptible Enterobacterales isolates frequently harbored carbapenemase and extended-spectrum β-lactamase (ESBL) genes, together with mutations in ftsI (encoding PBP3), ompK35, or siderophore receptor genes (cirA, tonB). DISCUSSION: Cefiderocol showed potent in vitro activity against CRGNB in Japan, including MBL producers. Disk diffusion correlated well with BMD results; however, confirmatory BMD testing should be considered when resistance is clinically suspected.

Despite the increasing number of reports on hypervirulent and extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae infections, data on the distribution of these pathogens in the community are limited. To address this knowledge gap, we investigated the carriage rates of K. pneumoniae complex in the stools of community-dwelling individuals in Japan. From 627 stool samples submitted to a commercial diagnostic laboratory, 407 Klebsiella strains were identified from 368 samples, corresponding to a colonization rate of 58.7%. Based on whole-genome sequencing, K. pneumoniae was the most prevalent species (n = 218, 53.6%), followed by Klebsiella variicola (n = 137, 33.7%). The detection rate of K. variicola was higher than previously reported in studies from other Asian countries. The overall distribution of sequence types (STs) was similar to those observed in previous studies of clinical isolates. However, hypervirulent K. pneumoniae clones, specifically ST23-K1 and ST412-K57, and ESBL-producing strains were rare, each accounting for less than 1% of the strains. These findings suggest that, while carriage of K. pneumoniae complex species is common in the community, healthcare settings may represent a more significant reservoir of hypervirulent and ESBL-producing K. pneumoniae strains in this epidemiological setting.IMPORTANCEKlebsiella pneumoniae complex species are bacteria that can cause serious infections, especially in hospital settings. Some types have become more dangerous because they are resistant to antibiotics or highly virulent. To better understand where these harmful clones come from, this study looked for Klebsiella species in healthy people living in the community in Japan. The results showed that these bacteria are commonly found in the gut, particularly K. pneumoniae and K. variicola. While some strains with traits linked to antibiotic resistance or severe infections were identified, they were rare. These findings suggest that most people carry Klebsiella strains as commensals and that the more dangerous forms of Klebsiella are likely spreading mainly in healthcare settings.

BACKGROUND: Carbapenem-resistant Gram-negative bacilli (CR-GNB) are a major public health threat, traditionally linked to hospital settings. However, infections are increasingly reported in the community, and the clinical distinctions between community-associated (CA) and healthcare-associated (HA) infections remain unclear. METHODS: We conducted a prospective multicenter study of hospitalized patients with CR-GNB infections across 13 Japanese tertiary hospitals between April 2019 and March 2024. Infections were categorized as CA, HA, or hospital-onset (HO) using standardized criteria. We compared patient demographics, microbiological findings, infection sites, and clinical outcomes based on the setting of onset. RESULTS: Among 425 patients, 43 had CA, 59 HA, and 323 HO infections. Pseudomonas aeruginosa was the predominant pathogen in all groups. Aeromonas species were more frequently associated with CA than HO cases (23.3% of CA vs 2.2% of HO cases), whereas Stenotrophomonas maltophilia was detected almost exclusively among HO cases. Hospital-onset infections were associated with longer median hospital stays compared with CA infections (68 vs 17 days) and a trend toward higher 30-day mortality (23.9% vs 9.5%). In contrast, HA infections demonstrated no significant differences from CA infections in either hospital length of stay (23 vs 17 days) or 30-day mortality rate (10.3% vs 9.5%). CONCLUSIONS: Community-associated CR-GNB infections are an emerging concern in Japan, showing distinct pathogen profiles and infection sites compared to HO cases. Importantly, HA infections resembled CA infections in terms of clinical characteristics and outcomes, suggesting a need to reexamine the clinical relevance of current HA classification criteria for guiding therapy and risk stratification.

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.