Exploring dangerous connections between Klebsiella pneumoniae biofilms and healthcare-associated infections

Healthcare-associated infections (HAI) are a huge public health concern,particularly when the etiological agents are multidrug resistant. The ability of bacteria to develop biofilm is a helpful skill, both to persist within hospital units and to increase antibiotic resistance. Although the links between antibiotic resistance, biofilms assembly and HAI are consensual, little is known about biofilms. Here, electron microscopy was adopted as a tool to investigate biofilm structures associated with increased antibiotic resistance. The K. pneumoniae strains investigated are able to assemble biofilms, albeit with different kinetics. The biofilm structure and the relative area fractions of bacteria and extracellular matrix depend on the particular strain, as well as the minimal inhibitory concentration (MIC) for the antibiotics. Increased values were found for bacteria organized in biofilms when compared to the respective planktonic forms, except for isolates Kp45 and Kp2948, the MIC values for which remained unchanged for fosfomycin. Altogether, these results showed that the emergence of antimicrobial resistance among bacteria responsible for HAI is a multifactorial phenomenon dependent on antibiotics and on bacteria/biofilm features

Molecular epidemiological analysis of Escherichia coli sequence type ST131 (O25:H4) and bla CTX-M-15among extended-spectrum-β- lactamase-producing E. coli from the United States, 2000 to 2009

Escherichia coli sequence type ST131 (from phylogenetic group B2), often carrying the extended-spectrum-β-lactamase (ESBL) gene bla , is an emerging globally disseminated pathogen that has received comparatively little attention in the United States. Accordingly, a convenience sample of 351 ESBL-producing E. coli isolates from 15 U.S. centers (collected in 2000 to 2009) underwent PCR-based phylotyping and detection of ST131 and bla . A total of 200 isolates, comprising 4 groups of 50 isolates each that were (i) bla negative non-ST131, (ii) bla positive non-ST131, (iii) bla negative ST131, or (iv) bla positive ST131, also underwent virulence genotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). Overall, 201 (57%) isolates exhibited bla , whereas 165 (47%) were ST131. ST131 accounted for 56% of bla -positive-versus 35% of bla -negative isolates (

Kinetic and Thermodynamic Characterization of the Bacterial Lectin FimH

One fundamental aim of drug discovery is the development of new molecular entities that have a considerably advantage over already existing therapies. Urinary tract infections (UTIs) urgently require an alternative to the conventional antibiotic therapy as resistance rates for antibiotics are increasing. The development of an anti-adhesive UTI treatment strategy with the bacterial lectin FimH as target is a promising approach to remedy such alarming tendencies. FimH is presented by uropathogenic E. coli (UPEC) strains on the tip of type 1 pili and mediates adhesion to mannosylated residues on the urothelium. This interaction prevents the clearance of UPECs during micturition and enables internalization of the pathogens by urothelial cells. Mannoside-derived FimH antagonists are under development and are considered as promising treatment option for UTIs. In contrast to antibiotics, FimH antagonists do not necessarily exert resistance mechanisms against drugs because they block the adhesion of bacteria to the urothelium without killing them or inhibiting their growth. ________ In the present thesis, FimH and its interaction with mannose-based antagonists were biophysically characterized. Additionally, new methodical approaches are introduced, which are relevant not only for a strategic development of FimH antagonists but also for drugs of other therapeutic areas. The following aspects were investigated: ________ Publication 2: The publication “KinITC – One method supports both thermo-dynamic and kinetic SARs” (Chemistry, 2018,24(49), 13049-13057) comments on kinITC-ETC, a new method based on ITC data to reveal the kinetic fingerprint of a drug–target interaction. In this study, kinITC-ETC was independently validated for the first time. Moreover, structural properties of FimH antagonists could be correlated with kinetic parameters of FimH–antagonist interactions. ________ Manuscript 1: The development of an off-rate screening approach is presented in the study “Off-rate screening by surface plasmon resonance – The search for promising lead structures targeting low-affinity FimH”. The method is subsequently applied to screen a mannose-based compound library against full-length FimH. The assay allows classification of structurally diverse FimH antagonist in order to spot chemical classes exhibiting long dissociative half-lives. ________ Publication 3: The lectin domain is conformationally rigid and needs the pilin domain for allosteric propagation. However, the crosstalk between allosteric sites within the lectin domain takes also place in the absence of the pilin domain as demonstrated in the publication “Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain – Engineering a minimalistic allosteric system” (J. Biol. Chem., 2018, 293(5), 1835-1849). Mutants of the isolated lectin domain, FimHLD R60P and V27C/L34C, exhibited a low-affinity state and mimic full-length FimH regarding its conformational transition upon mannoside binding. ________ Publication 4: The publication “Target-directed dynamic combinatorial chemistry: A study on potentials and pitfalls as exemplified on a bacterial target” (Chemistry, 2017, 23, 11570-11577) illustrates a target-directed dynamic combinatorial chemistry (tdDCC) approach employing reversible acylhydrazone formation with FimH full-length as target. Optimal sample preparation and data procession are discussed in detail. Finally, the results of the tdDCC assay were subsequently compared with the affinity of library constituents by SPR. ________ Publication 5: In the publication “Comparison of affinity ranking by target-directed dynamic combinatorial chemistry and surface plasmon resonance” larger FimH antagonist libraries were screened using the tdDCC method established in publication 3. The comparison of amplification rates of library substituents with respective binding affinities determined by SPR revealed a linear association. Furthermore, the hazardous acylhydrazone moiety could be replaced by various bioisosteres without changing the affinity of the parent compound. ________ Manuscript 2: The hydrogen bond network formed between mannose derivates and the CRD of FimH is extensively elucidated in the manuscript ”High-affinity carbohydrate–lectin interaction: How nature makes it possible”. Computational methods and structural prediction in combination with binding data revealed that the hydrogen bond network forms a unified whole. The removal of only a single hydroxyl group leads to a disruption of the cooperative interplay within the network and consequently results in a dramatic loss in binding affinity. ________ Manuscript 3: In the study “The tyrosine gate of the bacterial adhesion FimH – An evolutionary remnant paves the way for drug discovery”, ITC measurements demonstrated the influence of the tyrosine gate on binding affinity between FimH and natural ligands. While the tyrosine gate is exploited to form optimal hydrophobic interactions with aryl aglycones of synthetic FimH antagonists in order to increase their binding affinity, the tyrosine gate has only a marginal impact on the KD of natural ligands. In contrast to wild-type FimH, mutants that partially or completely lack the tyrosine gate exhibited a comparable binding affinity to dimannoside. ________ Publication 6: The publication “Improvement of aglycone π-stacking yields nanomolar to sub-nanomolar FimH antagonists” displays that fluorination of biphenyl mannosides further improved π-π stacking with the tyrosine gate, reaching nanomolar affinities with FimHFL and even picomolar affinities with FimHLD. It also could be shown that ligand binding to FimHFL occurs with a highly favorable enthalpic and a considerably unfavorable entropic contribution. ________ Publication 7: In the publication “Enhancing the enthalpic contribution of hydrogen bonds by solvent shielding” microcalorimetric studies of FimH could reveal that conformational adaptions of the binding site can establish a solvent-free cavity. Shielding the solvent results in a lower dielectric environment, in which the formation of hydrogen bonds has a considerable enthalpic contribution to the binding free energy. In the case of FimH approximately -13 kJ mol-1 for mannoside binding

Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics

This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs

DetectionOf genesResponsible for BiofilmsFormedby Klebsiella pneumoniaeandEsherichia coliand their effect on innate immunity

Biofilms as a major virulence factor of bacteria ,therelationship between bacteria persistence in the urinary tract and genes responsible for biofilms production was studied. The aim is to detect the presence,frequency of  the,fimA, fimH, mrkA  and  mrkD, genes  and biofilms production in two species isolated from UTI patients and their effect on some innate immunity aspects. Sixty five isolates of E.coli  and fifty of K.pneumoniae isolates were collected. All isolates were initially diagnosed  as genus Esherichiacoli andKlebsiella pneumonia. Final identification for the isolates was done by Vitek 2 compact system. The ability to form biofilm was carried out by using Tissue culture plate method (TCP).Adhesion average to epithelial cells of E.coli and K.pneumoniae was studied. Detection of genes were done to   by using PCR technique . Immunological experiments were done by using bactericidal activity and opsonization factor. UTI infection were more common in female than male. 75% (49/65) females, 25%(16/65)  males ofE.coli isolates and 88% (44/50) females,12%(6/50) males of K.pneumoniaeisolates. Patients ages in children between (2 months -18years) in UTI  caused by E.coli was 35%(23/65) while in k.pneumoniae was 74%(37/50),Ages between (18 years-50 years) in E.coli was 37%(24/65) while in K.pneumoniae was 18%(9/50) finally,Ages between (50 years-72years) in E.coli was 28%(18/65) while in K.pneumoniae was 8%(4/50).69% of E.coli isolates and 72% of k.pneumoniae isolates were higher biofilm producers. . Adhesion average to epithial cells of E.coli and K.pneumoniae was 42.9% and 44% respectively. Both E.coli and K.pneumoniae showed that 100% 0f the 12 isolate harbored the fimA, fimH, mrkA and mrkD genes.  The bactericidal average was higher in E.coli than K.pneumoniae  , P- value( 0.01)  for mice injected with fimbriae,  Opsonization  factor  was higher in E.coli than K.pneumoniae the P value (0.05) .                           Conclusion: A strong relationship between biofilms production and presence of genes of different species isolated from same clinical source that has effected on resistance of bacteria to innate immunity

Lectins as drug targets : functional, structural, and pharmacological insights into E-selectin and FimH

Lectins are carbohydrate-binding proteins found throughout nature in plants, viruses, pro- and eukaryotes. Their carbohydrate specificity as well as their biological functions are highly diverse. Lectins in humans primarily serve as cell-surface receptors and are mainly involved in the immunogenic processes. Bacterial lectins are often involved in conferring pathogenesis by binding to carbohydrates of the host. In this thesis, structural, functional, and pharmacological insights on the mammalian lectin E-selectin and the bacterial lectin FimH are given. Part E-selectin: E-selectin is a C-type lectin involved in leukocyte recruitment during inflammation by binding to the tetrasaccharide ligand sialyl Lewisx (sLex). It is involved in numerous diseases, e.g. asthma, psoriasis, stroke, rheumatoid arthritis or cancer metastasis. Targeting E-selectin with glycomimetic antagonists is therefore in the focus of drug discovery. The following aspects of E-selectin were investigated: -Publication 1: The thermodynamic driving forces of the interaction of E-selectin with sLex and glycomimetics thereof were investigated. We demonstrated that sLex binding is an entropy-driven process, which is an uncommon feature of carbohydrate-lectin interactions. -Manuscript 1: The co-crystallization of E-selectin with sLex or a more potent glycomimetics thereof revealed a previously unseen induced fit of the binding site involving alterations in the first two domains. We showed that this induced fit occurs in solution and discuss the physiological relevance. -Manuscript 2: A flexible and a pre-organized E-selectin antagonist were characterized for their kinetic and thermodynamic properties, which revealed an unexpected loss in entropy for the pre-organized antagonist. Co-crystallization with a series of antagonists revealed the reason for this behavior. -Publication 2: The single nucleotide polymorphism which leads to the S128R mutation in E-selectin has been correlated with an increased risk of developing various diseases. We investigated the binding behavior of this mutant and demonstrated that glycomimetics are efficacious in inhibiting E-selectin-S128R mediated binding. -Manuscript 3: Mice are able to express the N-glycolyl form of sLex, unlike humans. Therefore, the specificity of murine E-selectin might be altered. We investigated the binding specificity of murine E-selectin and evaluated the potency of antagonists designed for human E-selectin. We confirmed the efficacy of E-selectin antagonists towards murine E-selectin, thus demonstrating the validity of mouse models. Part FimH: The bacterial lectin FimH is presented by uropathogenic E. coli (UPEC) on the tip of type 1 pili and mediates the adhesion to mannosylated structure in the lower urinary tract. This interaction allows UPEC to colonize the bladder, the initial step in bladder infection. Mannoside-based FimH antagonists are under investigation as treatment for bladder infections. -Manuscript 4: The goal of a drug discovery program aimed to develop a treatment for urinary tract infections is to identify high-affinity, orally available, and safe FimH antagonists. Starting from the carboxylate substituted biphenyl alpha-D-mannopyranoside, affinity as well as the relevant pharmacokinetic parameters (solubility, permeability, renal excretion) could be substantially improved by a bioisosteric approach. -Manuscript 5: To comprehend and further develop potent FimH antagonists, structural data on ligand-protein interaction is essential. In this manuscript we present the X-ray co-crystal structures of FimH with three antagonist classes for which structural data were unavailable to date and provide an explanation for the observed entropy-enthalpy compensation by NMR. -Manuscript 6: Crystallographic studies of FimH with alkyl- or aryl-substituted alpha-D-mannopyranosides have demonstrated alternative binding poses with differing involvement of the residues Tyr48 and Tyr137 at the binding site entrance. Thermodynamic and molecular modeling analysis provided insights into the importance of the tyrosine-gate. -Manuscript 7: Several mutations of FimH are found in clinical isolates, which influence the binding phenotype of FimH by altering the interaction of the two FimH domains (lectin- and pilin domain). To date, FimH antagonists have never been tested on clinically relevant FimH variants. We demonstrated that antagonist affinity correlated with the binding behavior of different FimH variants

Uromodulin (Tamm–Horsfall protein): guardian of urinary and systemic homeostasis

Biology has taught us that a protein as abundantly made and conserved among species as Tamm–Horsfall protein (THP or uromodulin) cannot just be a waste product serving no particular purpose. However, for many researchers, THP is merely a nuisance during urine proteome profiling or exosome purification and for clinicians an enigmatic entity without clear disease implications. Thanks to recent human genetic and correlative studies and animal modeling, we now have a renewed appreciation of this highly prevalent protein in not only guarding urinary homeostasis, but also serving as a critical mediator in systemic inter-organ signaling. Beyond a mere barrier that lines the tubules, or a surrogate for nephron mass, mounting evidence suggests that THP is a multifunctional protein critical for modulating renal ion channel activity, salt/water balance, renal and systemic inflammatory response, intertubular communication, mineral crystallization and bacterial adhesion. Indeed, mutations in THP cause a group of inherited kidney diseases, and altered THP expression is associated with increased risks of urinary tract infection, kidney stone, hypertension, hyperuricemia and acute and chronic kidney diseases. Despite the recent surge of information surrounding THP’s physiological functions and disease involvement, our knowledge remains incomplete regarding how THP is normally regulated by external and intrinsic factors, how precisely THP deficiency leads to urinary and systemic pathophysiology and in what clinical settings THP can be used as a theranostic biomarker and a target for modulation to improve patient outcomes

Doctor of Philosophy

dissertationSepsis is a life-threatening systemic inflammatory condition that is characterized by a high degree of patient heterogeneity, making it notoriously difficult to diagnose and treat. Among the most common and lethal causes of sepsis are strains of Extraintestinal Pathogenic Escherichia coli (ExPEC). These genetically diverse pathogens are becoming increasingly problematic due to the rise of multidrug resistant strains. During sepsis, the infecting microbes are commonly viewed as generic inducers of inflammation, while the host background is considered the primary variable affecting disease progression and outcome. My doctoral research challenges this assumption and establishes a novel zebrafish embryo infection model to study the effects of ExPEC strain differences on the maladaptive immune responses that are induced during sepsis. Zebrafish embryos infected with ExPEC isolates display many of the key pathophysiological features seen in human septic patients, including dysregulated inflammatory responses (cytokine storms), tachycardia, and endothelial leakage. Mirroring what is seen in human patients, antibiotic therapy reduces bacterial titers in infected embryos and improves host survival rates, but is only effective within limited time frames, and surviving animals often develop lasting edema and other defects. Intriguingly, genetically distant ExPEC isolates stimulate markedly different host responses, including disparate levels of inflammatory (e.g., IL-1 β, TNF-α) and immunomodulatory (e.g., IL-10) mediators. These variances are attributable to differential activation of TLR5 by these strains, which vary in the levels and the serotypes of flagellin that they express. To examine the specific effects of IL-10 on disease progression, I created and begun to characterize engineered gene knockout and inducible transgenic zebrafish lines for this cytokine. Altogether, my graduate research establishes zebrafish as a relevant model for studying sepsis and highlights the ability of genetically distinct ExPEC isolates to induce divergent host responses independent of baseline host attributes and implicates bacterial flagellin as a key mediator of inflammation during sepsis

Investigation into mechanisms of biofilm formation by Klebsiella pneumoniae

Klebsiella pneumoniae is a Gram-negative opportunistic pathogen that normally causes nosocomial infections such as urinary tract infections, septicaemia, and respiratory tract infections. K. pneumoniae has become progressively resistant to the vast majority of antibiotics, and treatment of these particular infections is very challenging. At present, the emergence of antibiotic resistance is a widespread phenomenon that has been established as a major global healthcare threat. The ability of K. pneumoniae to form biofilms is known as one of the most important factors that contributes to the spread of this antibiotic-resistant bacterium. Due to that fact, understanding the mechanisms behind biofilm formation by K. pneumoniae is of the utmost importance. Numerous studies have shown that sub-inhibitory concentrations of antibiotics could induce biofilm formation by clinically important pathogens, for example, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. In this study, we report for the first time on the effect of sub-minimum inhibitory concentrations (sub-MICs) of gentamicin and ciprofloxacin on biofilm formation by K. pneumoniae. In addition, by using biofilm inhibition and disruption assays, we have also assessed the role of proteins in formation and composition of K. pneumoniae biofilms upon cultivation in nutritious and nutrient-poor media. As well as proteins, the role of polysaccharides and extracellular DNA was also determined. Furthermore, the role of type 1 fimbriae (T1F) in biofilm formation by K. pneumoniae was also determined. Transcription of fimA (the major subunit of T1F) is phase variable, as its promoter is on an invertible DNA element, fimswitch (fimS). We investigated the orientation of fimS in all K. pneumoniae strains used in this study, in planktonic and as biofilm cells, upon cultivation in tryptic soy broth (TSB) and artificial urine medium (AUM). Finally, we have also determined the role of outer membrane porins, OmpK35 and OmpK36 in biofilm formation by K. pneumoniae. We developed isogenic mutants of ∆ompK35 and ∆ompK36 strains. The capacity to form biofilms by the mutant strains compared to parental strains was examined in two different models, a microtitre plate model and a model using catheter pieces. Currently, we still lack a full understanding on the mechanisms of biofilm formation by K. pneumoniae which is important in order to develop new treatments to reduce the threat of biofilm-mediated infections by K. pneumoniae

Virulence potential of enterobacteriaceae isolated from neonatal enternal feeding tubes

In recent years, there has been a rise in the incidence of neonatal infections due to Enterobacteriaceae including Escherichia coli, Enterobacter, Klebsiella and Serratia spp. These are major causative agents in neonatal intensive care unit (NICU) infections. Neonates, especially those born with low birth weight (< 2000g), are fed via a nasogastric tube. Despite recent concerns over the microbiological safety of infant feeds, there has been no consideration that the nasogastric enteral feeding tube may act as a site for bacterial colonisation and act as a locus for infection. Therefore bacterial analysis of used feeding tubes is of importance with regard to identifying risk factors during neonatal enteral feeding. The aims of this study were to determine whether neonatal nasogastric enteral feeding tubes are colonised by opportunistic pathogens in the Enterobacteriaceae, and whether their presence was influenced by the feeding regime. In this research a collection 224 Enterobacteriaceae strains previously isolated from the enteral feeding tubes of neonates on intensive care units have been analysed. This study describes the use of DNA finger printing, via pulsed-field gel electrophoresis (PFGE), to determine if the same strains were isolated on different occasions from the NICUs. Therefore indicating whether certain strains have colonised the NICUs leading to increased exposure and risk to the neonates