Klebsiella pneumoniae exhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants

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

T. cruzi OligoC-TesT: A Simplified and Standardized Polymerase Chain Reaction Format for Diagnosis of Chagas Disease

Chagas disease (American trypanosomiasis) is caused by the protozoan parasite Trypanosoma cruzi and represents a major public health problem in Latin America. Furthermore, growing human population movements extend the disease distribution to regions outside the South American continent. Accurate diagnosis is crucial in patient care and in preventing transmission through blood transfusion, organ transplantation, or vertical transmission from mother to child. Routine diagnosis of Trypanosoma cruzi infection generally is based on detection of the host's antibodies against the parasite. However, antibody detection tests are liable to specificity problems and are of limited use in assessing treatment outcome and congenital infections. The introduction of the polymerase chain reaction (PCR) to amplify specific DNA sequences opened promising diagnostic perspectives. Despite its reported high sensitivity and specificity, broad use of the PCR technique in diagnosis of Chagas disease is hampered by its complexity and the lack of any standardization. We here present the development and evaluation of the T. cruzi OligoC-TesT, a simple and standardized dipstick format for detection of PCR amplified T. cruzi DNA. The new tool is an important step towards simplified and standardized molecular diagnosis of Chagas disease

TWIST1 Is Expressed in Colorectal Carcinomas and Predicts Patient Survival

TWIST1 is a transcription factor that belongs to the family of basic helix-loop-helix proteins involved in epithelial-to-mesenchymal transition and invasion processes. The TWIST1 protein possesses oncogenic, drug-resistant, angiogenic and invasive properties, and has been related with several human tumors and other pathologies. Colorectal cancer is one of the tumors in which TWIST1 is over-expressed, but its involvement in the clinical outcome of the disease is still unclear. We tested, by RT-PCR, the expression levels of TWIST1 in normal and tumor paired-sample tissues from a series of 151 colorectal cancer patients, in order to investigate its prognostic value as a tumor marker. TWIST1 expression was restricted to tumor tissues (86.1%) and correlated with lymph node metastasis (LNM). Adjusted analysis showed that the expression levels of TWIST1 correlated with overall survival (OS) and disease-free survival (DFS). Importantly, TWIST1 expression levels predicted OS specifically at stages I and II. Moreover, patients with stage II tumors and high TWIST1 levels showed even shorter survival than patients with stage III tumors. These results suggest that TWIST1 expression levels could be a tumor indicator in stage II patients and help select patients at greater risk of poor prognosis who might benefit from adjuvant chemotherapy

Investigation of One Health-related high-risk clonal lineages of multidrug resistant Escherichia coli and carbapenemase producing Klebsiella pneumoniae reveals key factors for their success

Antimicrobial resistance (AMR) is of paramount importance in the context of One Health, an integrated and unifying approach that aims to achieve a sustainable balance in the well-being of people, domestic and wild animals, plants, and their shared environments. Whenever bacteria become resistant to the therapeutic effects of antibiotics, they can cause infections that are difficult to treat effectively, increasing the risk of severe disease progression and death. Although AMR can develop naturally over time and is per se “ancient”, the excessive use of antibiotics in human and veterinary medicine over the past century has significantly accelerated its emergence and spread. Opportunistic Gram-negative enterobacteria, particularly Escherichia coli (E. coli ) and Klebsiella pneumoniae (K. pneumoniae) strains, increasingly exhibit resistance to multiple classes of clinically used antibiotics, thus presenting multidrug-resistant (MDR) phenotypes. To make matters worse, some of these strains combine multidrug resistance with high-level virulence, posing a threat to both immunocompromised and healthy individuals. Consequently, MDR E. coli and K. pneumoniae have been designated as high-risk pathogens by the World Health Organization, underscoring the urgent need for new antibiotic development. This thesis is motivated by the fact that only a limited number of international high-risk clonal E. coli and K. pneumoniae lineages stand out across all One Health dimensions and dominate the broad pool of MDR enterobacteria. While we only know little about the underlying drivers and contributing factors impacting their occurrence, emergence, and adaptation across different ecologies, this thesis employs a diverse range of bioinformatics and phenotypic approaches to identify the key factors important for the success of these lineages, also in rather under-explored settings. It includes three main components: (i) the analysis of genomic survey data of MDR E. coli isolates from ecologies in sub-Saharan Africa, (ii) the application of functional genomics and phenotyping techniques to characterize bacterial virulence and assess its clinical relevance in a food-borne E. coli strain, and (iii) the investigation of evolutionary pathways that promote the development of resistance to a novel drug combination and exploring compensatory mechanisms in a K. pneumoniae strain. To achieve these objectives, this research integrates genomics and transcriptomics with molecular biology and functional studies encompassing a comprehensive set of in vitro and in vivo virulence and resilience assays to explore MDR bacteria in-depth. We provide compelling evidence for the broad occurrence of successful high-risk clonal lineages in the One Health context and their circulation among clinics, wildlife, and food in international locations. In the first study, we isolated extended-spectrum β-lactamase (ESBL)-producing E. coli strains from houseflies collected from various wards at the University Teaching Hospital of Butare (Rwanda). In a follow-up study, we then examined in-depth the genomes of additional ESBL-producing E. coli from the same clinic and obtained from hospitalized patients, their caregivers, associated community members, and pets. The analyses revealed that the sample sets from this sub-Saharan African context consisted predominantly of globally recognized E. coli lineages, including sequence types (ST)131, ST167, ST410, and ST617. They play a pivotal role in the further dissemination and stabilization of AMR across diverse habitats within the One Health context. Moreover, our genomic results emphasize that these One Health-related high-risk clonal lineages exhibit the ability to successfully combine multidrug resistance with high-level bacterial virulence. To gain a more detailed understanding of the sophisticated interplay of virulence and AMR, we developed and refined a set of in vitro and in vivo methods for virulence phenotyping. These methodologies enabled us to characterize pathogens based on crucial clinical aspects such as biofilm formation, siderophore secretion, resistance to complement-mediated killing, and their capacity to cause mortality in Galleria mellonella larvae. By using a food-borne E. coli strain from an internationally recognized high-risk clonal lineage, we verified the remarkable combination of a MDR phenotype with clinically significant virulence properties, including synthesis of curli fibers and cellulose as part of biofilm formation, extensive secretion of siderophores, resilience against complement-containing human serum and pronounced mortality in the infection model. Nevertheless, the success of One Health-related high-risk clonal lineages does not rely solely on an “ideal” synergistic interplay between bacterial virulence and AMR. It also depends on their ability to rapidly mitigate the fitness costs associated with AMR acquisition, as these costs manifest in the form of reduced competitiveness and virulence in the absence of antibiotics. However, this is at odds with the observation of the global distribution of One Health-related high-risk clonal lineages across various One Health dimensions, even in environments with expectedly low selection pressures. To comprehensively address this, we conducted experimental evolution studies selecting for ceftazidime-avibactam-resistant mutants, which illuminated the rapid adaptations to changing environments. The adaptations and compensatory mechanisms were seemingly driven by major bacterial regulators, including the envelope stress response regulator RpoE on genomic and transcriptomic levels. In conclusion, the results of this thesis shed light on the fundamental principles that govern the character and interplay between AMR and bacterial virulence and advance our understanding of the contributors and drivers of successful MDR international high-risk clonal lineages in the One Health context. This is also important for effective and alternative intervention strategies to prospectively further address the global threat of AMR.Die Problematik der antimikrobiellen Resistenz (AMR) steht im Mittelpunkt des One Health Konzepts. Dieses Konzept umfasst einen ganzheitlichen Ansatz zur Sicherstellung eines nachhaltigen Gleichgewichts für das Wohlergehen von Menschen, domestizierten und wildlebenden Tieren, Pflanzen sowie ihrer gemeinsamen Umwelt. Wenn Bakterien gegen die therapeutische Wirkung von Antibiotika resistent werden, können sie Infektionen verursachen, die nur schwer wirksam zu behandeln sind und das Risiko eines schwerwiegenden Krankheitsverlaufs oder sogar des Todes erhöhen. Obwohl sich AMR im Laufe der Zeit natürlich entwickeln kann und daher an sich „vorzeitlich“ ist, hat der übermäßige Einsatz von Antibiotika in der Human- und Veterinärmedizin im letzten Jahrhundert ihr Auftreten und ihre Verbreitung erheblich beschleunigt. Opportunistische gramnegative Enterobakterien, darunter insbesondere Stämme von Escherichia coli (E. coli ) und Klebsiella pneumoniae (K. pneumoniae), weisen zunehmend Resistenzen gegen mehrere Klassen klinisch eingesetzter Antibiotika auf und zeigen somit multiresistente (MR) Phänotypen. Das Problem wird dadurch verschärft, dass einige dieser Stämme nicht nur MR, sondern auch hochvirulent sind, was sowohl für immungeschwächte als auch für gesunde Individuen ein ernsthaftes Risiko darstellt. Aus diesem Grund wurden MR E. coli und K. pneumoniae von der Weltgesundheitsorganisation als Hochrisikoerreger eingestuft, was den dringenden Bedarf an der Entwicklung neuer Antibiotika unterstreicht. Diese Dissertation ist durch die Tatsache motiviert, dass nur eine begrenzte Anzahl internationaler Hochrisiko-Klonlinien von E. coli und K. pneumoniae in allen Dimensionen des One-Health-Konzepts prävalent sind und die große Gruppe der MR Enterobakterien dominieren. Obwohl nur wenig über die zugrundeliegenden bakteriellen Triebkräfte und Faktoren bekannt ist, die ihr Auftreten, ihre Evolution und ihre Anpassung in verschiedenen Ökosystemen beeinflussen, verwendet diese Dissertation eine Vielzahl bioinformatischer und phänotypischer Methoden, um die Schlüsselfaktoren zu identifizieren, die für den Erfolg dieser klonalen Linien, auch in weniger erforschten Umgebungen, entscheidend sind. Die Arbeit gliedert sich daher in drei Hauptkomponenten: (i) die Analyse genomischer Daten von MR E. coli Stämmen aus Ökosystemen in Afrika südlich der Sahara, (ii) die Anwendung funktioneller Genomik und Phänotypisierung zur Charakterisierung der bakteriellen Virulenz und zur Bewertung der klinischen Relevanz am Beispiel eines E. coli Stammes aus rohem Fleisch und (iii) die Untersuchung der evolutionären Wege, die die Resistenzentwicklung gegenüber einer neuartigen Kombination von Wirkstoffen begünstigen, einschließlich der Untersuchung kompensatorischer Mechanismen in einem K. pneumoniae Stamm. Um diese Ziele zu erreichen, werden Genomik und Transkriptomik mit Molekularbiologie und funktionellen Studien kombiniert, einschließlich einer umfassenden Reihe von in vitro- und in vivo-Virulenz- und Resilienztests, um MR Bakterien im Detail zu untersuchen. Unsere Forschung präsentiert überzeugende Belege für das breitete Vorkommen von erfolgreichen Hochrisiko-Klonlinien im One-Health-Kontext, die sich in Krankenhäusern, der Tierwelt und Lebensmitteln an verschiedenen internationalen Standorten verbreiten. In unserer ersten Studie isolierten wir extended-spectrum β-lactamase (ESBL)-produzierende E. coli Stämme aus Stubenfliegen, die in verschiedenen Abteilungen des Universitätskrankenhauses in Butare (Ruanda) gesammelt wurden. In einer anschließenden Studie analysierten wir die Genome weiterer ESBL-produzierender E. coli Stämme aus derselben Klinik. Diese Stämme wurden sowohl von hospitalisierten Patienten, deren Betreuern sowie von Familienmitgliedern und Haustieren isoliert. Die Analysen zeigten, dass sich die Proben aus diesem subsaharisch-afrikanischen Kontext hauptsächlich aus international anerkannten E. coli Klonlinien zusammensetzten, einschließlich der Sequenztypen (ST)131, ST167, ST410 und ST617. Diese Klonlinien spielen daher eine entscheidende Rolle bei der weiteren Verbreitung und Konsolidierung von AMR in verschiedenen Habitaten des One-Health-Kontextes. Darüber hinaus unterstreichen unsere genomischen Ergebnisse, dass diese One-Health-bezogenen Hochrisiko-Klonlinien die Fähigkeit besitzen, erfolgreich Multiresistenz mit hoher bakterieller Virulenz zu kombinieren. Um dieses komplexe Zusammenspiel von Virulenz und AMR besser zu verstehen, haben wir eine Reihe von in vitro- und in vivo-Methoden zur Phänotypisierung der Virulenz entwickelt und optimiert. Diese Methoden ermöglichen die Charakterisierung von MR Bakterien hinsichtlich klinisch relevanter Aspekte, wie der Biofilmbildung, der Sekretion von Siderophoren, der Resistenz gegen Komplement-vermittelte Abtötung und ihrer Fähigkeit, Infektionen in Galleria mellonella Larven hervorzurufen. Unter Verwendung eines E. coli Stammes, der zu einer international anerkannten Hochrisiko-Klonlinie gehört und in Lebensmitteln gefunden wurde, konnten wir die bemerkenswerte Kombination eines MR Phänotyps mit klinisch bedeutsamen Virulenzeigenschaften, einschließlich der Synthese von Curli Fasern und Zellulose als Teil der Biofilmbildung, einer ausgeprägten Siderophor-Sekretion, der Widerstandsfähigkeit gegenüber menschlichem Serum und einer deutlichen Mortalität im Infektionsmodell bestätigt. Der Erfolg von One-Health-bezogenen Hochrisiko-Klonlinien beruht jedoch nicht nur auf einem „idealen“ synergistischen Zusammenspiel zwischen bakterieller Virulenz und AMR. Vielmehr resultiert er auch aus der Fähigkeit der MR Bakterien, die mit dem Erwerb von AMR verbundenen Fitnesskosten schnell zu reduzieren, um ihre Konkurrenzfähigkeit und Virulenz in Abwesenheit von Antibiotika aufrechtzuerhalten. Um dies umfassend zu untersuchen, haben wir experimentelle Evolutionsstudien durchgeführt, bei denen Mutanten selektiert wurden, die gegen Ceftazidim-Avibactam resistent sind. Diese Mutanten zeigten schnelle Anpassungen an veränderte Umweltbedingungen. Die Anpassungen und Kompensationsmechanismen scheinen auf genomischer und transkriptomischer Ebene durch wichtige bakterielle Regulatoren, wie den Regulator der membranständigen Stressantwort RpoE, gesteuert zu werden. Zusammenfassend tragen die Ergebnisse dieser Dissertation dazu bei, grundlegende Prinzipien aufzuklären, die den Charakter und die Interaktion von AMR und bakterieller Virulenz bestimmen. Gleichzeitig erweitern sie unser Verständnis der Faktoren und treibenden Kräfte erfolgreicher internationaler MR Hochrisiko-Klonlinien im One-Health-Kontext. Diese Erkenntnisse sind auch wichtig für die Entwicklung effektiver und alternativer Interventionsstrategien, um der globalen Bedrohung durch AMR proaktiv zu begegnen

Hypervirulent Klebsiella pneumoniae Sequence Type 420 with a Chromosomally Inserted Virulence Plasmid

Background: Klebsiella pneumoniae causes severe diseases including sepsis, pneumonia and wound infections and is differentiated into hypervirulent (hvKp) and classic (cKp) pathotypes. hvKp isolates are characterized clinically by invasive and multiple site infection and phenotypically in particular through hypermucoviscosity and increased siderophore production, enabled by the presence of the respective virulence genes, which are partly carried on plasmids. Methods: Here, we analyzed two K. pneumoniae isolates of a human patient that caused severe multiple site infection. By applying both genomic and phenotypic experiments and combining basic science with clinical approaches, we aimed at characterizing the clinical background as well as the two isolates in-depth. This also included bioinformatics analysis of a chromosomal virulence plasmid integration event. Results: Our genomic analysis revealed that the two isolates were clonal and belonged to sequence type 420, which is not only the first description of this K. pneumoniae subtype in Germany but also suggests belonging to the hvKp pathotype. The latter was supported by the clinical appearance and our phenotypic findings revealing increased siderophore production and hypermucoviscosity similar to an archetypical, hypervirulent K. pneumoniae strain. In addition, our in-depth bioinformatics analysis suggested the insertion of a hypervirulence plasmid in the bacterial chromosome, mediated by a new IS5 family sub-group IS903 insertion sequence designated ISKpn74. Conclusion: Our study contributes not only to the understanding of hvKp and the association between hypervirulence and clinical outcomes but reveals the chromosomal integration of a virulence plasmid, which might lead to tremendous public health implications

A Klebsiella pneumoniae ST307 outbreak clone from Germany demonstrates features of extensive drug resistance, hypermucoviscosity, and enhanced iron acquisition

Background Antibiotic-resistant Klebsiella pneumoniae are a major cause of hospital- and community-acquired infections, including sepsis, liver abscess, and pneumonia, driven mainly by the emergence of successful high-risk clonal lineages. The K. pneumoniae sequence type (ST) 307 lineage has appeared in several different parts of the world after first being described in Europe in 2008. From June to October 2019, we recorded an outbreak of an extensively drug-resistant ST307 lineage in four medical facilities in north-eastern Germany. Methods Here, we investigated these isolates and those from subsequent cases in the same facilities. We performed whole-genome sequencing to study phylogenetics, microevolution, and plasmid transmission, as well as phenotypic experiments including growth curves, hypermucoviscosity, siderophore secretion, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance for an in-depth characterization of this outbreak clone. Results Phylogenetics suggest a homogenous phylogram with several sub-clades containing either isolates from only one patient or isolates originating from different patients, suggesting inter-patient transmission. We identified three large resistance plasmids, carrying either NDM-1, CTX-M-15, or OXA-48, which K. pneumoniae ST307 likely donated to other K. pneumoniae isolates of different STs and even other bacterial species (e.g., Enterobacter cloacae) within the clinical settings. Several chromosomally and plasmid-encoded, hypervirulence-associated virulence factors (e.g., yersiniabactin, metabolite transporter, aerobactin, and heavy metal resistance genes) were identified in addition. While growth, biofilm formation, desiccation resilience, serum survival, and heavy metal resistance were comparable to several control strains, results from siderophore secretion and hypermucoviscosity experiments revealed superiority of the ST307 clone, similar to an archetypical, hypervirulent K. pneumoniae strain (hvKP1). Conclusions The combination of extensive drug resistance and virulence, partly conferred through a “mosaic” plasmid carrying both antibiotic resistance and hypervirulence-associated features, demonstrates serious public health implications.Peer Reviewe

Multidrug-Resistant High-Risk Escherichia coli and Klebsiella pneumoniae Clonal Lineages Occur in Black-Headed Gulls from Two Conservation Islands in Germany

Multidrug-resistant (MDR) Enterobacterales, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae, not only emerge in healthcare settings but also in other habitats, such as livestock and wildlife. The spread of these pathogens, which often combine resistance with high-level virulence, is a growing problem, as infections have become increasingly difficult to treat. Here, we investigated the occurrence of ESBL-producing E. coli and K. pneumoniae in fecal samples from two black-headed gull colonies breeding on two nature conservation islands in Western Pomerania, Germany. In addition to cloacal samples from adult birds (n = 211) and their nestlings (n = 99) during the 2021 breeding season, collective fecal samples (n = 29) were obtained. All samples were screened for ESBL producers, which were then subjected to whole-genome sequencing. We found a total of 12 ESBL-producing E. coli and K. pneumoniae consisting of 11 E. coli and 1 K. pneumoniae, and including the international high-risk E. coli sequence types (ST)131, ST38, and ST58. Eight of the investigated strains had a MDR genotype and carried a large repertoire of virulence-associated genes, including the pap operon, which is important for urinary tract infections. In addition, we identified many genes associated with adherence, biofilm formation, iron uptake, and toxin production. Finally, our analysis revealed the close phylogenetic relationship of ST38 strains with genomes originating from human sources, underlining their zoonotic and pathogenic character. This study highlights the importance of the One Health approach, and thus the interdependence between human and animal health and their surrounding environment

Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany

Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated in-depth an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021. Whole-genome sequence analysis revealed sequence type (ST)58, which has an internationally emerging high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Additionally, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens

Geno- and Phenotypic Characteristics of a Klebsiella pneumoniae ST20 Isolate with Unusual Colony Morphology

Klebsiella pneumoniae is a common member of the intestinal flora of vertebrates. In addition to opportunistic representatives, hypervirulent (hvKp) and antibiotic-resistant K. pneumoniae (ABR-Kp) occur. While ABR-Kp isolates often cause difficult-to-treat diseases due to limited therapeutic options, hvKp is a pathotype that can infect healthy individuals often leading to recurrent infection. Here, we investigated the clinical K. pneumoniae isolate PBIO3459 obtained from a blood sample, which showed an unusual colony morphology. By combining whole-genome and RNA sequencing with multiple in vitro and in vivo virulence-associated assays, we aimed to define the respective Klebsiella subtype and explore the unusual phenotypic appearance. We demonstrate that PBIO3459 belongs to sequence type (ST)20 and carries no acquired resistance genes, consistent with phenotypic susceptibility tests. In addition, the isolate showed low-level virulence, both at genetic and phenotypic levels. We thus suggest that PBIO3459 is an opportunistic (commensal) K. pneumoniae isolate. Genomic comparison of PBIO3459 with closely related ABR-Kp ST20 isolates revealed that they differed only in resistance genes. Finally, the unusual colony morphology was mainly associated with carbohydrate and amino acid transport and metabolism. In conclusion, our study reveals the characteristics of a Klebsiella sepsis isolate and suggests that opportunistic representatives likely acquire and accumulate antibiotic resistances that subsequently enable their emergence as ABR-Kp pathogens