Ângela Novais

Introduction: Carbapenemase-producing (CP) Klebsiella pneumoniae are a major public health concern in Portugal, where the population has been dominated by KPC-3-producing ST147-KL64. Following preliminary detection of an unexpectedly high proportion of ST15-KL19 infections after the COVID-19 pandemic, we investigated the recent expansion and clinical relevance of this sublineage. Methods: All CP K. pneumoniae isolates recovered from hospitalized patients between 2022 and 2023 ( n = 267) were characterized by antibiotic susceptibility testing and Fourier-transform infrared spectroscopy, wzi sequencing and multi-locus sequence typing. Representative ST15-KL19 isolates from a baseline (2019–2021) and the recent expansion (2022–2023) periods were selected for whole-genome sequencing and compared with publicly available global genomes to assess phylogenetic relatedness, temporal structure, and potential links to non-human reservoirs. Results: ST15-KL19 emerged as one of the most frequent CP K. pneumoniae sublineages, increasing from 23% to 35% and replacing ST147-KL64 over time. Genomic analyses identified two temporally distinct ST15-KL19 variants. Isolates from the recent expansion period (2022–2023) acquired bla _CTX–M–15 and other plasmid-mediated aminoglycoside resistance determinants, concurrently with an IncR plasmid replicon. Phylogenomic analyses revealed that both variants were already circulating in the region before 2019. Together with the high colonization rates at admission (64%), our data suggest repeated introductions from external sources rather than in-hospital evolution. Global comparisons supported regional and intercontinental dissemination from a common ancestor, though a direct foodborne origin could not be established. Conclusion: This study documents the recent expansion of a previously minor CP K. pneumoniae sublineage, highlighting how shifts in healthcare ecology can favor adaptive variants or repeated introductions. Continuous molecular surveillance is essential to detect such emerging lineages early and to inform infection control and antimicrobial stewardship strategies.

Klebsiella pneumoniae (Kp) is a major cause of bloodstream infections (BSI) globally. Yet, detailed characterization of invasive isolates, particularly from datasets not biased toward resistance, remains limited. This baseline study, conducted prior to the expansion of carbapenemase producers in Portugal, characterizes multidrug-resistant (MDR) and non-MDR Kp causing BSIs in a northern district hospital to elucidate resistance emergence and dissemination. A total of 174 Kp strains isolated from blood cultures of hospitalized patients (2016-2018) were identified by VITEK2 or MALDI-TOF MS. Antibiotic resistance phenotypes were determined by reference methods, β-lactamases screened by PCR/sequencing, and clonal sublineages identified by FT-IR spectroscopy, MLST and/or WGS, including for putative hypervirulent strains (hvKp). Isolates were categorized as MDR (49%) or non-MDR (51%), with MDR rates nearly double national reports. Most MDR-Kp (67%) produced CTX-M-15, 25% produced carbapenemases (mainly KPC-3 and/or OXA-48/CTX-M-15), and 8% lacked acquired β-lactamases. Half (53%) belonged to three high-risk sublineages (ST147-KL64, ST307-KL102, ST15-KL19), while non-MDR showed greater diversity, with ST14-KL2 most frequent (13%) and consistently resistant to amoxicillin-clavulanate. Several sublineages circulated across both cohorts (e.g., ST307-KL102, ST348-KL62), some with variable β-lactamase content (e.g., ST147-KL64, ST45-KL24). Two hvKp (1.1%) were identified (ST380-KL2, ST268-KL20). This baseline study reveals a high local burden of MDR-Kp driven by CTX-M-15 and KPC-3-producing high-risk sublineages, underscoring the importance of local surveillance beyond national reporting. High clonal diversity and variable β-lactamase content suggest heterogeneous acquisition and dissemination routes, with resistance often preceding clonal expansion. Though rare, hvKp detection underscores the need for continued surveillance and multicenter monitoring.

Expansion of carbapenemase-producing Klebsiella pneumoniae (CP- Kp ) is driven by nosocomial dissemination, and effective infection control depends on timely and reliable typing data. Here, we evaluated our previously developed Fourier-transform infrared spectroscopy (FT-IR) with attenuated total reflectance (ATR) workflow for real-time typing of Kp capsular (KL)-types and lineages to support infection control. FT-IR spectra were acquired from Columbia agar with 5% sheep blood cultures of all CP- Kp infection isolates (n=136) from hospitalized patients at a northern Portugal hospital (April 2022 – March 2023), and analyzed using automated machine-learning (ML) classification models. Typing results were confirmed by wzi sequencing, MLST and/or WGS. FT-IR typing on Columbia agar plates showed 73% sensitivity, 79% specificity and 74% accuracy. Our method correctly typed 94% of typeable isolates (78/83), from which 87% were comunicated in Kp typing. Three Kp lineages (ST147-KL64, ST15-KL19, ST268-KL20) represented 74% of the sample, with ST268-KL20 causing an outbreak in Neonatal Intensive Care unit, quickly recognized by FT-IR enabling immediate infection control measures. Epidemiological links between patiens were mostly found on medical, surgical and urology units, using EpiLinx software. Most isolates (98%) produced KPC-3. Our FT-IR ATR ML-based typing workflow demonstrated high performance standards in real-time and high adaptability to clonal dynamics. The unprecedent time-to-response (same day of species identification) represents an opportunity to implement timely and effective infection control measures. Importance: This study represents the first prospective and real-time evaluation of FT-IR spectroscopy to type multidrug resistant Klebsiella pneumoniae to support surveillance and infection control. We demonstrate a high sensitivity, specificity and accuracy of a previously developed workflow that allows precise identification of K. pneumoniae lineages. The adaptability to changes in clonal dynamics and bacterial typing in <24h offer significant advantages in both high- and low-income countries for a timely infection control and improvement of antimicrobial resistance management.

Whole genome sequencing (WGS) has become a vital tool in clinical microbiology, playing an important role in outbreak investigations, molecular surveillance, and identification of bacterial species, resistance mechanisms and virulence factors. However, the complexity of WGS data presents challenges in interpretation and reporting, requiring tailored strategies to enhance efficiency and impact. This study explores the diverse needs of key stakeholders in healthcare, including clinical management, laboratory work, public surveillance and epidemiology, infection prevention and control, and academic research, regarding WGS-based reporting of clinically relevant bacterial species. In order to determine preferences regarding WGS reports, human-centered design approach was employed, involving an online survey and a subsequent workshop with stakeholders. The survey gathered responses from 64 participants representing the above mentioned healthcare sectors across geographical regions. Key findings include the identification of barriers related to data accessibility, integration with patient records, and the complexity of interpreting WGS results. As the participants designed their ideal report using nine pre-defined sections of a typical WGS report, differences in needs regarding report structure and content across stakeholders became evident. The workshop discussions further highlighted the need to feature critical findings and quality metrics prominently in reports, as well as the demand for flexible report designs. Commonalities were observed across stakeholder-specific reporting templates, such as the uniform ranking of certain report sections, but preferences regarding the depth of content within these sections varied. Using these findings, we suggest stakeholder-specific structures which should be considered when designing customized reporting templates. In conclusion, this study underscores the importance of tailoring WGS-based reports of clinically relevant bacteria to meet the distinct needs of diverse healthcare stakeholders. The evolving landscape of digital reporting increases the opportunities with respect to WGS reporting and its utility in managing infectious diseases and public health surveillance.

Carbapenemase-producing Enterobacterales are increasingly being recognized in nosocomial infections. The performance of a flow cytometry-based rapid assay for their detection and differentiation was evaluated. This is a disruptive phenotypic technology, phenotypic and growth-independent, that searches for the lesions produced by drugs acting on cells after a short incubation time. Overall, 180 Gram-negative bacteria were studied, and results were compared with those obtained molecularly by PCR and phenotypically by ‘KPC, MBL and OXA-48 Confirm Kit’. This phenotypic method was used as reference for comparison purposes. Susceptibility to carbapenems (imipenem, meropenem, and ertapenem) was determined by standard broth microdilution. Overall, 112 isolates (62.2%) were carbapenemase producers, 41 KPCs, 36 MβLs, and 31 OXA-48, and 4 strains were KPC + MβL co-producers. Sixty-eight isolates were carbapenemase-negative. The percentage of agreement, sensitivity, and specificity were calculated according to ISO 20776-2:2021. The FASTinov assay showed 97.7% agreement with the reference method for carbapenemase detection. Discrepant flow cytometry results were obtained in four isolates compared with both reference and PCR results. The sensitivity and specificity of this new technology were 95.3% and 98.5%, respectively, for KPCs, 97.6% and 99.3% for MβLs, and 96.9% and 98% for OXA-48 detection. In conclusion, we describe a rapid flow cytometry assay with high accuracy for carbapenemase detection and the differentiation of various carbapenemases, which should impact clinical microbiology laboratories and patient management.

We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance.

The rise of antibiotic resistance in the food chain is influenced by the use of antimicrobial agents, such as antibiotics, metals, and biocides, throughout the entire farm-to-fork continuum. Besides, non-clinical reservoirs potentially contribute to the transmission of critical pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae . However, limited knowledge exists about the population structure and genomic diversity of K. pneumoniae circulating in conventional poultry production. We conducted a comprehensive characterization of K. pneumoniae across the whole chicken production chain (flocks/environment/meat, 2019-2022), exploring factors beyond antibiotics, like copper and quaternary ammonium compounds (QACs). Clonal diversity and adaptive features of K. pneumoniae were characterized through cultural, molecular (FT-IR), and whole-genome-sequencing (WGS) approaches. All except one flock were positive for K. pneumoniae with a significant increase (p < 0.05) from early to pre-slaughter stages, most persisting in chicken meat batches. Colistin-resistant K. pneumoniae rates were low (4%), while most samples carried MDR strains (67%) and copper-tolerant isolates (63%; sil + pco clusters; MIC _CuSO4 ≥16mM), particularly at pre-slaughter. Benzalkonium chloride consistently exhibited activity in K. pneumoniae (MIC/MBC range=4-64mg/L) from diverse and representative strains independently of the presence/absence of genes linked to QACs tolerance. A polyclonal K. pneumoniae population, discriminated by FT-IR and WGS, included various lineages dispersed throughout the chicken’s lifecycle at the farm (ST29-KL124, ST11-KL106, ST15-KL19, ST1228-KL38), until the meat (ST1-KL19, ST11-KL111, ST6405-KL109, and ST6406-CG147-KL111), or over years (ST631-49 KL109, ST6651-KL107, ST6406-CG147-KL111). Notably, some lineages were identical to those from human clinical isolates. WGS also revealed F-type multireplicon plasmids carrying sil + pco (copper) co-located with qacE Δ1± qacF (QACs) and antibiotic resistance genes like those disseminated in humans. In conclusion, chicken farms and their derived meat are significant reservoirs for diverse K. pneumoniae clones enriched in antibiotic resistance and metal tolerance genes, some exhibiting genetic similarities with human clinical strains. Further research is imperative to unravel the factors influencing K. pneumoniae persistence and dissemination within poultry production, contributing to improved food safety risk management. This study underscores the significance of understanding the interplay between antimicrobial control strategies and non-clinical sources to effectively address the spread of antimicrobial resistance.

The spread of bacteria resistant to last-resort antibiotics such as colistin throughout the food chain is a serious concern for public health. The poultry sector has responded by restricting colistin use and exploring alternative trace metals/copper feed supplements.