Background: Prevention of nosocomial infections continues to be crucial to ensure patient safety and improve healthcare outcomes. In this regard, surface contamination plays an important role in the undetected transmission of nosocomial pathogens as a continuous, sporadic event or in the context of outbreaks. However, the impact of reducing bacterial contamination through copper-coated surfaces remains controversial. Methods: A pilot study was set up in a blinded, randomized controlled design to elucidate the antimicrobial activity of door handles coated with a copper-nickel alloy. Twelve doors in a specialized department of tumor and revision surgery of a German orthopedic hospital were randomly selected to install visually indistinguishable stainless-steel door handles, either without coating (control group, n=6) or with an alloy coating consisting of 30% copper and 70% nickel (study group, n=6). Patients, all involved personnel and investigators were blinded with regard to the assignment of door handles. Door handles were sampled for viable microorganisms at 24 h after disinfection by (i) consecutive use of wet and dry swabs and (ii) contact agar slides. Bacterial growth was detected and bacteria were identified using MALDI-TOF mass spectrometry. In addition, contamination kinetics of door handles were determined by ATP measurement at time points 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after disinfection. Each technique was used on three subsequent days. Results: Using swab method, the mean total number of colony forming units (cfu) of control and copper-nickel alloy surfaces was 2.14 cfu/cm2 and 0.67 cfu/cm2, respectively, yielding a difference of 68.7% (p=0.27). Bacterial counts from contact agar slide samples resulted in 0.86 cfu/cm2 on control and 0.6 cfu/cm2 on coated door handles which equals a difference of 30.2% (p=0.31). ATP bioluminescence measured over three subsequent days from coated door handles showed a decreased bioburden by 70.8%, 23.1%, 55.5%, 79.7%, 45.9%, 56.0%, and 68.3% of relative light units compared to control door handles at time points 0 h (before disinfection), 0 h (after disinfection), 1 h, 2 h, 4 h, 8 h, and 12 h, respectively. Statistically significant differences (p<0.05) were obtained for time points 4 h and 12 h. Conclusion: Our data indicate a trend of reduced bacterial and overall bioburden on copper-nickel-coated door handles. Further, larger randomized controlled trials are warranted to investigate the influence of copper-coated surfaces on the prevention of hospital-acquired infections.

Citrobacter freundii is a nosocomial pathogen increasingly associated with multidrug resistance and hospital outbreaks. Despite its growing clinical relevance, no standardized core genome multilocus sequence typing (cgMLST) scheme has been available for high-resolution epidemiological analyses. Here, we developed and validated a robust cgMLST scheme for C. freundii comprising 3,250 target loci based on a curated data set of 825 globally distributed genomes representing extensive sequence type and geographic diversity. Validation against published outbreak data sets from hospitals in Finland and Belgium, as well as environmental and patient isolates from two German university hospitals, proved that the scheme possesses high target gene coverage (median 99.6%) and strong discriminatory power. Additionally, we developed a combined cgMLST scheme for C. freundii, C. portucalensis, C. braakii, and C. europaeus, based on 2,307 shared target loci and target gene coverages of ≥99.7%. This scheme proved suitable for cross-species outbreak analysis. Our analyses revealed environments, such as sinks, shower drains, and toilets, as likely reservoirs where Citrobacter species may persist in hospital settings. These findings suggest that environmental sources could play a significant role in transmission events involving patients, while allele-based cluster analyses indicated that direct patient-to-patient transmission was rare. Given the increasing prevalence of multidrug-resistant (MDR) Citrobacter strains, the level of discrimination achieved by these newly developed cgMLST schemes underscores the importance of accurate species identification and environmental screening in understanding the transmission dynamics of opportunistic healthcare-associated pathogens. Ultimately, this makes them valuable tools for genomic surveillance, outbreak investigation, and infection prevention. IMPORTANCE:Accurate identification and high-resolution typing of multidrug-resistant bacteria are essential for understanding their transmission dynamics in hospitals, particularly in light of the global spread of resistant strains and the role of environmental reservoirs. The newly developed cgMLST schemes presented here provide standardized, portable tools for both local and global scientific and clinical communities to conduct fine-scale genomic epidemiology across four Citrobacter species. These schemes support detailed outbreak reconstruction, source attribution, and cross-hospital comparisons, capabilities that are critical in an era of increasing antimicrobial resistance and international patient movement. By enabling consistent, species-specific surveillance and comparative analyses, cgMLST enhances infection control and public health responses, facilitating early detection and targeted intervention.

BACKGROUND:Infections caused by hypervirulent Klebsiella pneumoniae (hvKp) are often characterised by severe, metastatic and relapsing infections. Initially described in Asia, this pathotype has now expanded worldwide. Convergent strains combining hypervirulence with multidrug resistance additionally aggravate the situation. However, only sparse data are available on the occurrence of hvKp in European countries. Therefore, this study investigated the prevalence of hvKp in a tertiary medical centre in Germany. METHODS:K. pneumoniae isolates were prospectively collected from clinical specimens obtained at the University Medicine Greifswald from June 1st to August 31st, 2022. Only the first isolate from each patient was considered, while screening samples were excluded. All isolates were phenotypically characterised for virulence and subjected to whole genome sequencing (WGS). RESULTS:A total of 122 isolates were included, which ranged from largely antibiotic-susceptible to multidrug-resistant phenotypes, with only one isolate carrying a carbapenemase gene (blaOXA-181). Phenotypic assays showed heterogeneous results, with only one isolate demonstrating concomitant positivity in both the string test and mucoid-staining plate. WGS revealed 81 different sequence types, including high-risk clonal lineages. Four isolates carried typical genetic markers associated with hypervirulence and were largely antibiotic-susceptible. In the Galleria mellonella infection model, these four isolates showed higher larval mortality compared to two control carbapenem-resistant classical K. pneumoniae strains. Convergent strains were not found. CONCLUSION:Our findings revealed that 3.3 % (4 out of 122) of isolates were classified as hvKp. This prevalence appears clinically relevant due to the notoriously aggressive course of infections caused by this pathotype.

Abscesses, erysipelas, or uncomplicated skin and soft tissue infections (phlegmons) are common soft tissue infections encountered by dermatologists. Their definition, diagnosis, and treatment are summarized here based on the updated German-language guidelines. Cutaneous abscesses are encapsulated, pus-filled cavities which, depending on their location in deep or superficial skin-layers, cause solid to fluctuating, more or less erythematous, painful swelling under usually intact epidermis. Superficial and mature abscesses reveal visible collection of pus. Primary and often sufficient therapy consists of incision and drainage. In the presence of complicating factors, an antibiotic targeting Staphylococcus (S.) aureus is additionally recommended. Erysipelas, caused by streptococci, is characterized by bright-red, often shiny erythema with sometimes arched margins, which is initially accompanied by constitutional symptoms such as shivers or fever. The treatment of choice is penicillin. An uncomplicated soft tissue infection is usually caused by S. aureus and is characterized by a livid red erythema with slight edema and blurred margins. Constitutional symptoms do not generally occur and sometimes only in the course of infection. They are sufficiently treated with, e.g., oral cefadroxil or intravenous cefazolin. Complicated soft tissue infections are extensive infections of soft tissue due to certain comorbidities, such as severe diabetes, immunosuppression, liver or kidney failure or to complicating factors such as burns or injuries with foreign bodies. The spectrum of causative bacteria is broader and must be considered for calculated antibiotic therapy. This review is intended to provide targeted and successful antibiotic therapy and to reduce adverse effects and development of resistant bacteria.

This study (n = 668) investigated the effect of vaccination on SARS-CoV-2 infection among individuals with type 2 diabetes mellitus (T2DM), cancer, and smoking in a prospective cohort study in Northern Germany. Participants from the SHIP-COVID cohort were followed from October 2020 to October 2022 using repeated questionnaires and serological tests. Infection was defined by anti-nucleocapsid (anti-NCP) antibodies’ detection, self-reported Real Time Polymerase Chain Reaction (RT-PCR) or rapid antigen test. Vaccine efficacy was evaluated using risk differences (RD), risk ratios (RR), p-values, and 95% confidence intervals. Infection risk decreased with more vaccine doses; the 3rd (RR = 0.68) and 4th (RR = 0.29) doses offered significant protection. Both sexes showed protection with 3rd and 4th doses (RR = 0.67 and 0.20 in women and 0.68 and 0.39 in men, respectively). Logistic regression showed higher infection odds in individuals with T2DM (14%) and cancer (68% in SHIP-START-3 dataset and 45% in SHIP-START-4 dataset), while current smokers had 47% and 45% lower odds than non-smokers in SHIP-START-3 and SHIP-START-4 datasets respectively. No significant interactions were found between vaccination and T2DM, cancer, or smoking.

Despite recent advances in error rate reduction, until recently, Oxford Nanopore Technologies (ONT) sequences lacked the accuracy required for fine-scale bacterial genomic analysis. Here, recent software improvements of ONT and the ONT-core-genome multilocus sequence typing (cgMLST)-Polisher within the SeqSphere+ software were evaluated. We used short-read (Illumina) and long-read ONT sequences of 80 multidrug-resistant organisms (MDROs) for benchmarking. Illumina reads were de novo assembled using SKESA. For ONT, Dorado Super Accurate (SUP) model v.4.3 or v.5.0 basecalled reads were assembled with Flye and then polished with Medaka v.1.12 m4.3 or Medaka v.2.0 bacterial methylation model. In addition, the ONT-cgMLST-Polisher was run over all assemblies. The "ground truth" (GT) hybrid assemblies were created using Hybracter v.0.10.0. Sixteen isolates from four species out of the original 80 isolates were sent to six laboratories for a ring trial. The 80 MDROs basecalled with SUP m4.3 had an average cgMLST allele distance (AD) to the GT of 4.94 with Medaka v.1.12 and 1.78 with Medaka v.2.0, respectively. After further polishing the Medaka v.2.0 data with the ONT-cgMLST-Polisher, the AD dropped to 0.09. Using data basecalled with SUP m5.0 with Medaka v.2.0 further reduced the AD significantly to 0.04. While the ring trial data basecalled with Dorado SUP m4.3 showed more variability and insufficient results for some samples, model 5.0 data resulted in average ADs of 0.36 and 0.17 without and with the ONT-cgMLST-Polisher, respectively. In conclusion, recent ONT Dorado and Medaka models combined with the ONT-cgMLST-Polisher improved ONT sequencing accuracy and made it sufficiently reproducible for genomic surveillance of bacteria.IMPORTANCEONT sequencing methodology is especially attractive for small and medium-sized laboratories due to its relatively low capital investment and price per sample consumable costs. However, until recently, it lacked accuracy and reproducibility for bacterial genomic genotyping. Here, we present an evaluation of the most recent ONT bioinformatic (basecalling and polishing of consensus) improvements and a new ONT-cgMLST-Polisher tool. We demonstrate that by applying those procedures, ONT whole-genome genotyping-based surveillance of bacteria is finally accurate and reproducible enough for routine application even in small laboratories.

Klebsiella pneumoniae, an important opportunistic pathogen, is traditionally classified into classic and hypervirulent pathotypes. Convergent strains combining antimicrobial resistance (AMR) and hypervirulence have emerged globally, posing a significant health challenge. In this study, we investigated potential in-host evolution and morphotypic variation among consecutive K. pneumoniae ST147 isolates from a single patient. As described in a previous study, the isolates displayed distinct morphotypes-small white, normal white, gray, and gray/dry (g/d) colonies. In this study, we now present new genomic and phenotypic analyses, which suggest the acquisition of AMR and virulence genes through plasmid gain. The early isolates carried fewer plasmids, resulting in low resistance and virulence, while later isolates acquired a hybrid IncFIB-IncHI1B plasmid encoding different resistance and aerobactin genes, significantly increasing their geno- and phenotypic AMR and virulence potential. Chromosomal integration of this plasmid in one isolate stabilized the acquired traits. Disruptions in the K locus, mediated by insertion sequences, were linked to the gray and g/d morphotypes, impairing capsule biosynthesis. Uronic acid assays confirmed reduced capsule production in these isolates. In contrast, small colony variants showed significant transcriptomic changes, including upregulation of capsule biosynthesis, iron uptake pathways, and AMR genes, suggesting persistence through altered metabolism. Our findings suggest in-host microevolution of K. pneumoniae ST147 from a classic to a convergent pathotype and highlight the genomic and transcriptomic adaptations underlying morphotypic diversity, providing new insights into the pathogen's adaptability and persistence in certain environments.IMPORTANCEKlebsiella pneumoniae is an important bacterial pathogen that can cause severe infections, particularly in healthcare settings. Traditionally, strains are classified as either drug-resistant or highly virulent, but recent strains combining both features have led to hard-to-treat infections. In this study, we investigated the potential in-host evolution of K. pneumoniae ST147 isolates from a single patient, revealing key genomic and phenotypic changes driving their adaptability. Over time, isolates acquired additional plasmids carrying antimicrobial resistance and virulence genes, including a hybrid plasmid integrated into the bacterial chromosome, stabilizing its beneficial traits. In addition, morphotypic diversity emerged, linked to genomic disruptions in capsule biosynthesis genes (K locus). While capsule-deficient morphotypes displayed structural changes, small colony variants exhibited transcriptomic adaptations to persist under stress. This work underscores the dynamic evolutionary capacity of K. pneumoniae adapting within the host, providing critical insights into its persistence, resilience, and the challenges posed by emerging convergent strains in clinical settings.

IntroductionThe Klebsiella pneumoniae (KP) species complex (KpSC) comprises KP as the predominant species, and six other taxa including two subspecies each of Klebsiella variicola (KV) and Klebsiella quasipneumoniae (KQ), all capable of causing clinical infections and often challenging to differentiate. Among these, KP is by far the most clinically significant, with the emergence of multidrug-resistant and hypervirulent strains leading to severe infections and limited treatment options, underscoring the need to understand the genomic features of KP.MethodsThis study compared globally disseminated KP lineages with less abundant KV strains in synthetic human urine (SHU) across multiple omics levels to identify characteristics differentiating these closely related species. Moreover, a large genomic dataset of over 6,000 publicly available genomes of KP, KV, and KQ was constructed for comparisons to other members of the KpSC.ResultsAmong eight clinical KP strains representing four different sequence types (STs), we identified 107 genes comprising the KP-specific core genome, while these genes were absent from two selected KV genomes. Transcriptome and proteome analyses in SHU revealed different regulatory patterns between KP and KV strains, with metabolic responses playing a pivotal role. A total of 193 genes specific to the investigated KP STs were identified, exhibiting differential expression at the transcriptomic and/or proteomic levels. Comparison to the genomic dataset highlighted genes adaptively regulated or uniquely present in KP genomes. For example, certain genes for citrate metabolism are uniquely upregulated in KP and a gene cluster for the cellobiose phosphotransferase system, previously linked to bacterial virulence and biofilm formation, was found exclusively in KP.DiscussionOur study underscores the metabolic flexibility of KP strains in response to specific environmental conditions, potentially contributing to opportunistic pathogenicity. We identified markers enriched in KP STs, providing a foundation for future investigations including their relevance for diagnostics.

BackgroundIntracellular invasion and persistence of Staphylococcus aureus can lead to chronic infection and is an effective strategy for the pathogen to evade the host immune response and antibiotic therapy. Selective ubiquitination of bacterial surfaces via E3 ubiquitin ligases is a mechanism by which host cells combat intracellular bacteria and target them for autophagosomal degradation. However, knowledge of the E3 ligases involved in intracellular recognition of S. aureus is still very limited.MethodsWe studied A549 lung epithelial cells during S. aureus infection, focusing on the role of the E3 ligase leucine rich repeat and sterile alpha motif containing 1 (LRSAM1). We used the CRISPR-Cas9 system to generate LRSAM1-deficient A549 cells and monitored intracellular bacterial survival, activation of host cellular signalling pathways related to cytokine production, and host cell death during S. aureus infection.ResultsIn LRSAM1-deficient host cells we observed a significant increase in intracellular bacterial load, which was accompanied by an increased host cell death and elevated secretion of the pro-inflammatory cytokine IL-6. Despite induced selective autophagy, LRSAM1 knockout host cells were incapable of lowering and eliminating the pathogen, which seems to be caused by the reduced ubiquitination of the bacterial surface.ConclusionThe results indicate a significant role of LRSAM1 in the clearance of intracellular S. aureus. This contributes to a deeper understanding of the host cellular responses to S. aureus infection and will facilitate the development of novel therapeutic strategies to combat intracellularly persistent S. aureus.

The benefit of antibiotic treatment (ABT) for patients with moderate COVID-19 is unclear and overtreatment poses the risk of adverse effects such as Clostridioides difficile infection and antibiotic resistance. This multi-center study compares health status improvement between patients with and without ABT at hospital admission. Between March 2020 and May 2023, hospitalized adults with confirmed SARS-CoV-2 infection were recruited from the German National Pandemic Cohort Network (NAPKON), which includes patients from various hospitals across Germany. The study population included patients with moderate or severe COVID-19 at baseline. The primary objective was to compare health improvement or decline after two weeks between patients who received ABT at baseline and those who did not in the moderate COVID-19 population. The statistical analysis adjusted for confounders such as gender, age, vaccination status, clinical condition, and comorbidities. The severe COVID-19 population was investigated as a secondary objective. A total of 1,317 patients (median age 59 years; 38

OBJECTIVES:Campylobacter species represent one of the leading causes of human foodborne infections, including gastroenteritis and bloody diarrhoea. Overuse of antibiotics in veterinary, agriculture, and humans has led to an increase in multidrug antimicrobial resistance (AMR). Fluoroquinolones and macrolides resistant Campylobacters are WHO and CDC priority pathogens, with fluoroquinolone resistance doubling in the past 20 years, complicating treatment. METHODS:Published studies relating to AMR and associated molecular mechanisms in both Campylobacter jejuni (C. jejuni) and C. coli from animals, humans and environment (1981-2024), were retrieved from PubMed and Google Scholar using relevant keywords. In addition, genomic analyses of publicly available C. jejuni and C. coli genomes along with multilocus sequence typing results from the PubMLST database were used to analyse these AMR determinants and their phylogenomic relationships. Review articles were excluded from the analyses. RESULTS:A total of 429 research papers were reviewed to get insights into multidrug resistance in C. jejuni and C. coli. Fluroquinolone resistance has been predominantly associated with international travel. The gyrA subunits were associated with ecological niches and overall, it is suggestive that C. coli might be the donor. A positive synergism was observed between cmeA gene expression and quinolone resistance. Additionally, the results speculated the possibility of horizontal gene transfers in chromosomal resistance clusters between C. coli and C. jejuni. CONCLUSIONS:This review indicated significant concern of multidrug resistance in C. jejuni and C. coli. This requires continent-wide surveillance and research for standard practices to achieve effective antimicrobial stewardship.

In this study, we characterized a Klebsiella pneumoniae strain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes in Enterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 isolates with different resisto- and morpho-types were distinguished from the patient’s urine and tissue samples. Whole genome sequencing revealed that all isolates were clonally identical belonging to the K. pneumoniae high-risk sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic “hyper-splitting” in a K. pneumoniae SCV and its potential clinical significance.

Plasma-activated water (PAW) generated from tap water has gained attention as a disinfectant when used directly in its pure form. Little is known about the application of PAW for bacterial inactivation in aqueous environments because its use in fluids results in dilutions. We investigated the effect of PAW in aqueous suspensions simulating such dilutions, and we focused on the minimal addition of PAW volumes to bacterial aqueous suspensions still resulting in high inactivation rates. The antimicrobial effect was highly dependent on the activation of PAW. An increase in activation power from 90 to 100 W resulted in a greater microbial reduction with an identical 10 min activation time. The susceptibility to PAW dilutions was analyzed in detail regarding nine Gram-negative species out of Enterobacterales and other waterborne microorganisms as well as four Gram-positive species present in two different matrices, in saline and in tap water, at high concentrations simulating massive contamination situations. For this purpose, the PAW activation setting of 90 W and 30 min was defined in order to be able to differentiate the limitations of inactivation in individual bacterial species. The Gram-negatives in saline demonstrated susceptibility when one volume unit of PAW was added. However, twice the PAW volume was necessary for inactivation when bacteria were present in tap water. Gram-positive microorganisms were more robust, indicated by prolonged contact times before inactivation. Our results indicate that PAW can be used for bacterial decontamination processes in aqueous environments when added in surplus. Optimized activation settings such as electric power to generate PAW and the contact times to the samples increase the effect of the inactivation a wide range of bacteria, regardless of their resistance profiles.

Metals and metalloids are used as weapons for predatory feeding by unicellular eukaryotes on prokaryotes. This review emphasizes the role of metal(loid) bioavailability over the course of Earth's history, coupled with eukaryogenesis and the evolution of the mitochondrion to trace the emergence and use of the metal(loid) prey-killing phagosome as a feeding strategy. Members of the genera Acanthamoeba and Dictyostelium use metals such as zinc (Zn) and copper (Cu), and possibly metalloids, to kill their bacterial prey after phagocytosis. We provide a potential timeline on when these capacities first evolved and how they correlate with perceived changes in metal(loid) bioavailability through Earth's history. The origin of phagotrophic eukaryotes must have postdated the Great Oxidation Event (GOE) in agreement with redox-dependent modification of metal(loid) bioavailability for phagotrophic poisoning. However, this predatory mechanism is predicted to have evolved much later - closer to the origin of the multicellular metazoans and the evolutionary development of the immune systems.

Viral infections can lead to platelet activation and hemostatic complications. However, the extent to which platelet reactivity remains altered after convalescence, contributing to long-term health impairments as observed after COVID-19 is not yet fully understood. Therefore, we conducted a cohort study (DRKS00025217) to determine platelet function in individuals convalesced from mild COVID-19. Assays were performed ex vivo with blood from convalescents at 2-15 weeks and 6-10 months after convalescence, focusing on platelet aggregation, activation markers, and thrombin formation. In addition, two other potentially relevant factors for platelet function were examined: the immunomodulatory mediator sphingosine-1-phosphate (S1P) and the platelet expression of the transporter MRP4 (ABCC4). Our findings indicate that robust platelet functions, including platelet aggregation determined by light transmission aggregometry, and thrombin formation, were not altered in convalescents compared to matched control individuals. However, an elevation in subtle platelet activation markers, such as P-selectin surface expression and activation of glycoprotein IIb/IIIa, was observed 2-15 weeks after convalescence. This was accompanied by an increased expression of MRP4 in platelets and significantly elevated levels of S1P in platelet-poor plasma. Our findings suggest increased platelet sensitization and a pro-inflammatory state even after convalescence from mild COVID-19, pointing toward MRP4 and S1P as associated factors.

This case-report focuses on a 23-year-old soldier suffering from a fracture-related hip joint infection (FRI) due to extensively drug-resistant Klebsiella pneumoniae and S. epidermidis. The patient underwent multiple septic revision surgeries including the removal of remaining shrapnel accompanied by last-resort antimicrobial therapy with cefiderocol and colistin. Additionally, the surgeries included repeated tissue sampling for microbiological and histopathological analysis. An antibiotic-loaded cemented filler containing cefiderocol was used to improve local antimicrobial therapy. The biopsies prior to and during hip replacement surgery confirmed successful microbe eradication. Hip arthroplasty restored hip joint function and significantly improved patient's quality of life. The utilization of a trabecular metal shell and a meta-diaphyseally anchored cementless hip stem ensured secure implant fixation and early patient mobilisation. An adjusted biofilm active oral antimicrobial therapy after arthroplasty intervention was continued to prevent early periprosthetic joint infection. This case emphasizes the difficulties of managing FRI and multidrug-resistant pathogens. It contributes valuable insight into navigating complex orthopedic cases while ensuring successful hip arthroplasty outcomes. In conclusion, early interdisciplinary collaboration, appropriate antimicrobial therapy along with tailored surgical interventions are crucial for managing such complex cases successfully.

Klebsiella pneumoniae , an important opportunistic pathogen, has long been categorized into two distinct pathotypes: the often multidrug-resistant classic (cKp) and the highly virulent hypervirulent (hvKp). However, a recent global trend has witnessed the emergence of convergent strains, seamlessly combining antimicrobial resistance with hypervirulence. Our study delved into a series of K. pneumoniae isolates sourced from the same patient, all belonging to the international, high-risk clonal lineage of sequence type 147. As reported in a previous study, these isolates exhibited diverse morphotypes on blood agar, ranging from small white to normal-sized white, grey, or grey and dry (g/d) colonies.Through an interplay of omics and phenotypic experiments, we unraveled the intricate mechanisms governing these distinct colony morphologies and their implications on bacterial virulence and resilience. While the earlier isolates demonstrated modest levels of resistance and virulence, their later counterparts showed significantly heightened levels, attributed to the acquisition of additional plasmids. Bioinformatics analysis unveiled a chromosomal insertion of a hybrid plasmid in one isolate, marking an unprecedented in-host microevolution from the classic to the convergent pathotype.All morphotypes exhibited positive insertion sequences around or within the K loci, with the grey or g/d phenotypes arising from impaired K loci. Despite lower serum resistance, these morphotypes demonstrated superior adhesion to human epithelial cells. Interestingly, while capsule-deficient strains are conventionally associated with decreased virulence, our isolates displayed high mortality rates in the Galleria mellonella infection model.In conclusion, our findings not only provide unprecedented insights into in-host microevolution within a patient, transitioning from the classic to the convergent pathotype, but also contribute significantly to the understanding of the diverse morphotypes exhibited by K. pneumoniae .### Competing Interest StatementThe authors have declared no competing interest.

BackgroundConvergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307.MethodsHypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics.ResultsOur results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways.ConclusionThis study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.