Sohei Harada

UNLABELLED: Phenotypic identification of hypervirulent Klebsiella pneumoniae in routine laboratories remains challenging. We evaluated tellurite resistance and selected carbohydrate fermentation tests as phenotypic tools for screening hypervirulent strains and improving lineage identification. A total of 193 K. pneumoniae species complex strains with available whole-genome sequencing data were analyzed, including K. pneumoniae (n = 152), K. variicola (n = 29), and K. quasipneumoniae (n = 12). Hypervirulent strains were defined as those carrying ≥4 of 5 virulence-associated genes (iucA, iroB, peg-344, rmpA, and rmpA2). A total of 68 strains (35.2%) were classified as hypervirulent, and 74 (38.3%) carried ter genes. Hypervirulent strains were strongly associated with ter gene carriage, and the tellurite-resistant phenotype on MacConkey agar containing 4 μg/mL potassium tellurite closely matched ter gene carriage. Among tellurite-resistant strains, adonitol non-fermentation was useful for excluding non-hypervirulent K. variicola. Using combined tellurite resistance and adonitol fermentation, the assay identified hypervirulent strains with a sensitivity of 86.8%, specificity of 89.6%, and accuracy of 88.6%, all higher than the string test. All 27 ST23 strains showed dulcitol fermentation and l-sorbose non-fermentation. Adonitol fermentation improved screening performance, whereas dulcitol and l-sorbose fermentation patterns supported presumptive identification of prototypical hypervirulent lineages. These findings support the potential utility of this culture-based approach for phenotypic screening of hypervirulent K. pneumoniae in clinical microbiology laboratories. IMPORTANCE: Hypervirulent Klebsiella pneumoniae is associated with severe invasive infections, but practical methods for early recognition in routine clinical laboratories remain limited. Leveraging the strong association between hypervirulence genotype and ter gene carriage, we developed a simple culture-based screening approach for the early presumptive identification of hypervirulent K. pneumoniae. This approach, based on combined tellurite resistance and adonitol fermentation, shows higher sensitivity and specificity than the string test for identifying hypervirulent strains. Additionally, dulcitol and l-sorbose fermentation patterns support presumptive identification of prototypical hypervirulent lineages, including ST23. These phenotypic assays may expedite the identification of hypervirulent K. pneumoniae and selected hypervirulent lineages in clinical microbiology laboratories.

BACKGROUND: The current clinical paradigm recommends appropriate blood culture collection based on the patient's severity and suspected diagnosis. Beyond providing microbiological diagnosis, blood cultures can facilitate the de-escalation of broad-spectrum antibiotics. Nonetheless, blood culture collection practices upon prescription of broad-spectrum antibiotics remain largely unexplored. METHODS: A retrospective observational study was conducted at a tertiary care academic hospital in Tokyo, Japan. Patients who required hospitalization and underwent empiric piperacillin/tazobactam prescription on day of admission from 2016/4/1 to 2017/12/31 were included. Patients ≤18 years old, who finished piperacillin/tazobactam on or before day 1, and duplicates were excluded. A total of 250 patients were randomly selected to assess the frequency and factors associated with blood culture collection. RESULTS: Blood culture collection was fulfilled in 163 patients (65.2%). The use of immunosuppressive agents (odds ratio [OR], 3.47; confidence interval [CI], 1.43-9.43), systemic inflammatory response syndrome criteria ≥2 (OR, 4.42; CI, 2.22-9.23) were associated with blood culture collection. On the contrary, intraabdominal infection (OR, 0.28; CI 0.11-0.65), and surgical specialty (OR, 0.33; CI, 0.18-0.61) were associated with withholding of blood culture collection. CONCLUSIONS: Withholding of blood culture collection was observed prior to empiric piperacillin/tazobactam prescription in one-third of patients warranting hospitalization. Surgical specialty was associated with potential missed opportunities for blood culture collection. Tailored implementation strategies may optimize blood culture collection practices among different specialties.

Carbapenems remain essential for treating serious infections caused by drug-resistant Gram-negative bacteria because of their broad-spectrum activities and favourable safety profiles. However, the emergence of carbapenemase-producing Enterobacterales, which produce enzymes that efficiently hydrolyse β-lactams including carbapenems, continues to undermine their clinical utility. Although new antibiotics such as ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, and cefiderocol have expanded therapeutic options, their effectiveness varies substantially across different carbapenemase families. Carbapenemases produced by Enterobacterales include serine β-lactamases (Ambler classes A and D) and metallo-β-lactamases (MBLs; Ambler class B), each with distinct substrate and inhibitor profiles. Clinically relevant MBLs-including imipenemase (IMP), New Delhi MBL (NDM), and Verona integron-encoded MBL (VIM) variants-show markedly different biochemical properties and inhibitor susceptibilities. Despite their clinical relevance, optimal treatment strategies for infections caused by IMP-producing Enterobacterales remain poorly defined. The unique reactivity of IMP-type MBLs to inhibitors differs from that of other MBLs such as NDMs or VIMs, underscoring the need for tailored therapeutic approaches. In this Personal View, we summarise current evidence and, drawing on Japan's experience as an endemic setting for IMP producers, outline key scientific, clinical, and public health challenges that should be addressed globally to develop effective, evidence-based treatment strategies for IMP-producing Enterobacterales infections.

OBJECTIVE: Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) have a poor prognosis, particularly in immunocompromised patients. While carbapenemase production is the primary resistance mechanism in CRKP, other mechanisms may contribute synergistically to carbapenem resistance. Here, we describe a case in which extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae, initially susceptible to carbapenems, acquired carbapenem resistance without carbapenemase production following ceftolozane-tazobactam exposure. METHODS: Whole-genome sequencing was performed on ESBL-producing K. pneumoniae strains detected during the clinical course to investigate the relationship between the genetic characteristics and antimicrobial susceptibility, particularly to ceftolozane-tazobactam and carbapenems. Changes in the transcription levels of the beta-lactamase genes were also analyzed using reverse transcription quantitative PCR (RT-qPCR). RESULTS: Active surveillance stool culture before cord blood transplantation (CBT) identified colonization with an ESBL-producing K. pneumoniae strain. Febrile neutropenia occurred on day 7 after CBT and was treated empirically with ceftolozane-tazobactam. Fatal bacteremia developed on day 30 after CBT due to a carbapenemase-negative isolate resistant to both ceftolozane-tazobactam and carbapenems. Whole-genome sequencing analysis revealed that K. pneumoniae ST307 strains harboring blaCTX-M-15, blaSHV-28, and blaTEM-1B acquired a nonsense mutation in ompK36 following ceftolozane-tazobactam exposure. RT-qPCR analysis documented a significant increase in blaSHV-28 transcription after ceftolozane-tazobactam exposure. CONCLUSIONS: This case demonstrates that K. pneumoniae without carbapenemase production can acquire carbapenem resistance through a combined resistance mechanisms following exposure to non-carbapenem antibiotics. Antimicrobial stewardship must be implemented comprehensively, not focused solely on specific antibiotics.