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dr hab. Beata Krawczyk
Employment
- Associate professor at Katedra Biotechnologii i Mikrobiologii
Publications
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total: 80
Catalog Publications
Year 2024
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Biodiversity of microorganisms in the Baltic Sea: the power of novel methods in the identification of marine microbes
PublicationUntil recently, the data on the diversity of the entire microbial community from the Baltic Sea were relatively rare and very scarce. However, modern molecular methods have provided new insights into this field with interesting results. They can be summarized as follows. (i) Although low salinity causes a reduction in the biodiversity of multicellular species relative to the populations of the North–East Atlantic, no such reduction...
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Chronic Rhinosinusitis—Microbiological Etiology, Potential Genetic Markers, and Diagnosis
PublicationChronic rhinosinusitis (CRS) is a significant public health problem. Bacterial colonization and impaired mucociliary clearance play a significant role in the inflammatory process. Several inflammatory pathways and host defense elements are altered in CRS, which may contribute to observed differences in the microbiome. To date, researching CRS has been difficult due to limited access to the studied tissue and a lack of available...
Year 2023
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Asymptomatic Bacteriuria in Kidney Transplant Recipients—A Narrative Review
PublicationUrinary tract infections (UTIs) are the most prevalent complications in kidney transplant (KTx) recipients. The most frequent finding in this group of patients is asymptomatic bacteriuria (ASB). Here, we provide an overview of the available evidence regarding ASB in KTx recipients, including its etiopathology, clinical impact and management. There is a growing body of evidence from clinical trials that screening for and treating...
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Optical method supported by machine learning for urinary tract infection detection and urosepsis risk assessment
PublicationThe study presents an optical method supported by machine learning for discriminating urinary tract infections from an infection capable of causing urosepsis. The method comprises spectra of spectroscopy measurement of artificial urine samples with bacteria from solid cultures of clinical E. coli strains. To provide a reliable classification of results assistance of 27 algorithms was tested. We proved that is possible to obtain...
Year 2022
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Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats
PublicationWild birds can be colonized by bacteria, which are often resistant to antibiotics and have various virulence profiles. The aim of this study was to analyze antibiotic resistance mechanisms and virulence profiles in relation to the phylogenetic group of E. coli strains that were isolated from the GI tract of wildfowl. Out of 241 faecal samples, presence of E. coli resistant to a cephalosporin (ESBL/AmpC) was estimated for 33 isolates...
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BEXUS 30 Stardust – Preliminary Results of Investigation of Microbes in the Stratosphere
PublicationThe stratospheric microbiome has been investigated several times using the methods of classical microbiology. In this experiment, we have combined them with some novel approaches.. Diversification of methods has helped better describe the collected microorganisms. The results of the experiment may help better understand the mechanisms of survivability of microorganisms in stratospheric conditions.
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Breastfeeding as a regulating factor of the development of the intestinal microbiome in the early stages of life
PublicationSince the first bacterial inhabitants of the human gastrointestinal tract were identified, a lot of research into the study of the human microbiome and its effects on health has been conducted. Currently, it is accepted that humans have a symbiotic relationship with the gut microbiome, though the specifics of this relationship are not well understood. The microbiome of neonates constantly changes and appears to influence many facets...
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INVESTIGATION OF THE EFFECT OF THE ROCKET'S SUBORBITAL FLIGHT ON BIOFILM, ENZYMES AND BIOSYNTHESIS ON AUTONOMOUS, MODULAR AND SCALABLE PLATFORM FOR CONDUCTING EXPERIMENTS OF AN ASTROBIOTECHNOLOGICAL NATURE
PublicationWith new incentives for human space exploration, biotechnological experiments in orbit became imperative.Answering this need, we build an autonomous, modular, and scalable platform that enables those experiments onrockets. We called it AMBER. The aim of the payload of the R6 suborbital rocket is to perform an experiment tostudy the influence of rocket flights on biofilm, molecular...
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Investigation of the Effect of the Rocket's Suborbital Flight on Biofilm, Enzymes and Biosynthesis on Autonomous, Modular and Scalable Platforms For Conducting Experiments of an Astrobiotechnological Nature
PublicationWith new incentives for human space exploration, biotechnological experiments in orbit became imperative. Answering this need, we build an autonomous, modular, and scalable platform that enables those experiments on rockets. We called it AMBER. The aim of the payload of the R6 suborbital rocket is to perform an experiment to study the influence of rocket flights on biofilm, molecular biology enzymes and biosynthesis using our platform....
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May Staphylococcus lugdunensis Be an Etiological Factor of Chronic Maxillary Sinuses Infection?
PublicationStaphylococcus lugdunensis is an opportunistic pathogen found in the healthy human skin microbiome bacterial community that is able to cause infections of diverse localization, manifestation, and course, including laryngological infections, such as necrotizing sinusitis. Chronic maxillary sinusitis is a disease present in up to one third of European and American populations, and its etiology is not fully described. Within this...
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Metody metagenomiczne w badaniu mikrobiomu jelitowego człowieka
PublicationMikroorganizmy zamieszkujące ciało człowieka to nie tylko komensale i groźne patogeny, ale także organizmy symbiotyczne pełniące kluczową rolę w zachowaniu homeostazy gospodarza. Badania metagenomiczne wykorzystujące techniki sekwen-cjonowania nowej generacji i metody bioinformatyczne pozwalają na dogłębne po-znanie mikrobiomu ludzkiego i poszukiwanie jego związku ze stanem zdrowia czło-wieka. Celem pracy jest omówienie różnych...
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Stardust - Investigation of Microbes in the Stratosphere
PublicationThe stratospheric microbiome has been investigated several times using the methods of classical microbiology. In this experiment, we have combined them with some novel approaches including whole- metagenome amplification, Maldi TOF mass spectrometry and Sanger DNA sequencing. The results of the experiment may provide the scientists with knowledge about the mechanisms of survivability of microorganisms in stratospheric conditions...
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Urinary Tract Infections Caused by K. pneumoniae in Kidney Transplant Recipients – Epidemiology, Virulence and Antibiotic Resistance
PublicationUrinary tract infections are the most common complication in kidney transplant recipients, possibly resulting in the deterioration of a long-term kidney allograft function and an increased risk of recipient’s death. K. pneumoniae has emerged as one of the most prevalent etiologic agents in the context of recurrent urinary tract infections, especially with multidrug resistant strains. This paper discusses the epidemiology and risk...
Year 2021
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Evaluating the antibacterial activity of muramyl dipeptide derivatives, retro-tuftsin derivatives, and anthraquinone oligopeptides against a range of pathogenic bacteria
PublicationSearch for new and efficient antibiotic is crucial because of microbial drug resistance and problems with side effects of the administered medication. In this study, we evaluate the in vitro microbiological activity of muramyl dipeptide derivatives, retro-tuftsin derivatives (i.e., tuftsin with reversed amino acid sequences), and combinations of retro-tuftsin derivatives with substituted anthraquinones. The potency of the investigated...
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Genetic Background and Antibiotic Resistance Profiles of K. pneumoniae NDM-1 Strains Isolated from UTI, ABU, and the GI Tract, from One Hospital in Poland, in Relation to Strains Nationally and Worldwide
PublicationIn recent years, there has been an observed increase in infections caused by carbapenem-resistant Klebsiella pneumonia (Kp) strains. The aim of this study was the phenotypic and genotypic analysis of eight K. pneumoniae NDM (Kp NDM) isolates, recovered in Poland during the years 2016 and 2018 from seven patients with urinary tract infections (UTIs), asymptomatic bacteriuria (ABU), or colonization of the gut. PCR melting profile...
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Siderofory bakteryjne – rola w patogenezie i potencjał wykorzystania w diagnostyce
PublicationDo prawidłowego funkcjonowania i wzrostu organizmów żywych zarówno ludzi, jak i bakterii niezbędne jest żelazo. Mikroelement ten występuje na dwóch różnych stopniach utlenienia: jako jon żelazawy (Fe2+) rozpuszczalny w roztworach fizjolo-gicznych oraz jako jon żelazowy (Fe3+), który nie tworzy roztworów wodnych [1]. Dzięki swoim właściwościom oksydo-redukcyjnym, żelazo wpływa na poprawne funkcjonowanie bardzo licznej grupy enzymów,...
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The Many Faces of Enterococcus spp.—Commensal, Probiotic and Opportunistic Pathogen
PublicationEnterococcus spp. are Gram-positive, facultative, anaerobic cocci, which are found in the intestinal flora and, less frequently, in the vagina or mouth. Enterococcus faecalis and Enterococcus faecium are the most common species found in humans. As commensals, enterococci colonize the digestive system and participate in the modulation of the immune system in humans and animals. For many years reference enterococcal strains have...
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X-ray and UV Radiation Damage of dsDNA/Protein Complexes
PublicationRadiation and photodynamic therapies are used for cancer treatment by targeting DNA. However, efficiency is limited due to physico-chemical processes and the insensitivity of native nucleobases to damage. Thus, incorporation of radio- and photosensitizers into these therapies should increase both efficacy and the yield of DNA damage. To date, studies of sensitization processes have been performed on simple model systems, e.g.,...
Year 2020
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Escherichia coli Strains with Virulent Factors Typical for Uropathogens were Isolated from Sinuses from Patients with Chronic Rhinosinusitis—Case Report
PublicationEscherichia coli were isolated from three patients with chronic rhinosinusitis (CRS) by intraoperative sinus tissue biopsy. Taking into account the unusual replicative niche and previous treatment failures, it was decided to focus on the virulence and drug resistance of these bacteria. The strains turned out to be multi-sensitive, but the rich virulence factors profile of bacteria typical for phylogenetic group B2 deserved attention....
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Linezolid-resistant Enterococcus faecium strains isolated from one hospital in Poland –commensals or hospital-adapted pathogens?
PublicationOne of the most pressing problems of enterococci infections is occurring resistance to linezolid, which is an antibiotic used in the treatment of infections caused by vancomycin-resistant strains (VRE). The main objective of our research was to investigate the relationship of 19 linezolid-resistant E. faecium isolates from 18 patients hospitalized at Clinical Hospital in Gdansk (Poland). One of the LZDREF was isolated in 2003...
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The New Klebsiella pneumoniae ST152 Variants with Hypermucoviscous Phenotype Isolated from Renal Transplant Recipients with Asymptomatic Bacteriuria-Genetic Characteristics by WGS.
PublicationKlebsiella pneumoniae (Kp) is one of the most important etiological factors of urinary tract infections in renal transplant (RTx) recipients. We described the antimicrobial susceptibility phenotypes and genomic features of two hypermucoviscous (HM) Kp isolates recovered from RTx recipients with asymptomatic bacteriuria (ABU). Using whole genome sequencing (WGS) data, we showed that the strains belong to the ST152 lineage with the...
Year 2019
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Charakterystyka oporności Klebsiella pneumoniae New Delhi na karbapenemy
PublicationBakterie Klebsiella pneumoniae New Delhi, posiadające gen oporności na karbapenemy (blaNDM) zostały po raz pierwszy wyizolowane w 2008 roku w Szwecji od pacjenta wcześniej hospitalizowanego w Indiach. Bakterie te szybko rozprzestrzeniają się po całym świecie, a od 2011 roku występują również w Polsce. Stanowią one wysokie ryzyko epidemiologiczne, ponieważ gen oporności na karbapenemy przekazywany jest pomiędzy bakteriami poprzez...
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Host and pathogen factors in Klebsiella pneumoniae upper urinary tract infections in renal transplant patients
PublicationPurpose . To analyse the role of virulence factors (VFs) and host in Klebsiella pneumoniae upper urinary tract infections (UTIs) in renal transplant (RTx) recipients. Methodology. Clinical and demographic data were registered prospectively. Phylogenetic background of K. pneumoniae isolates was analysed by PCR melting profiles (MP) and the following VFs genes: fimH-1, uge, kpn, ycfM, mrkD, rmpA, magA, hlyA, cnf-1, irp-1, irp-2,...
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Patomechanizm zakażeń dróg moczowych wywoływanych przez uropatogenne szczepy E. coli
PublicationZakażenia układu moczowego (ZUM) to jedne z najczęstszych i najbardziej powszechnych infekcji bakteryjnych, dotykających każdego roku nawet 150 milionów ludzi na świecie. Problem z tego typu infekcjami wynika z przewlekłości choroby, nawrotów i wzrastającej lekooporności powodujących je patogenów. Uropatogenne szczepy E. coli (UPEC) są głównym czynnikiem przyczynowym ZUM. Bakterie tej grupy zawierają wiele czynników adhezyjnych...
Year 2018
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Fusion of DNA-binding domain of Pyrococcus furiosus ligase with TaqStoffel DNA polymerase as a useful tool in PCR with difficult targets
PublicationThe DNA coding sequence of TaqStoffel polymer- ase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the Taq DNA polymerase from a relatively low processive...
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Intra-operative biopsy in chronic sinusitis detects pathogenic Escherichia coli that carry fimG/H, fyuA and agn43 genes coding biofilm formation
PublicationThe aim of this study was to investigate whether or not surgical biopsy of sinus tissue in chronic sinusitis, not responsive to treatment, would detect E. coli. We intended to evaluate E. coli virulence genes, therefore dispute the causal role of such an unusual microorganism in chronic sinusitis, as well as consider effective pathogen-targeted therapy. Patients with E. coli isolated by intra-operative puncture biopsy were included...
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Molecular identification and genotyping of Staphylococci: genus, species, strains, colnes, lineages, and interspecies exchanges
PublicationStaphylococci are increasingly recognized as etiological agents of many opportunistic human and animal infections, emphasizing the need for a rapid and accurate identification, even to a genotypical level of these bacteria. In the recent years, there has been a significant progress in typing and phylogenetic study of Staphylococcus species. Here, we describe molecular methods used in taxonomy as well as staphylococci characterization....
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Synthesis and antimicrobial activity of amino acid and peptide derivatives of mycophenolic acid
PublicationThe series of 16 novel amino acid and peptide mycophenolic acid (MPA) derivatives was obtained as potential antibacterial agents. Coupling of MPA with respective amines was optimized with condensing reagents such as EDCI/DMAP and T3P/TEA. Amino acid analogs were received both as methyl esters and also with the free carboxylic group. The biological activity of the products was tested on five references bacterial strains: Klebsiella...
Year 2017
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A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification
PublicationCandida sp. have been responsible for an increasing number of infections, especially in patients with immunodeficiency. Species specific differentiation of the Candida sp. is difficult in routine diagnosis. This identification can have a highly significant association in therapy and prophylaxis. This work has shown a new application of terminal restriction fragment length polymorphism (t-RFLP) method in molecular identification...
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Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization
PublicationDNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the...
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Modified DNA polymerases for PCR troubleshooting
PublicationPCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase...
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Modyfikacje polimeraz DNA jako rozwiązania problemów w reakcjach PCR
PublicationReakcja PCR już od wielu lat jest podstawowym narzędziem stosowanym w biotechnologii molekularnej. Nadal napotykamy jednak liczne problemy podczas jej przeprowadzania związane z trudnymi matrycami, obecnością inhibitorów itp. Optymalizacja reakcji PCR jest więc niezbędna w celu poprawy wyników. Jednym z rozwiązań tych problemów może być modyfikowanie polimeraz DNA. W powyższym artykule opisano problemy i rozwiązania zależne polimerazy...
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Recurrent bowel-blood translocations of Escherichia coli with the unique virulence characteristics over three-year period in the patient with acute myeloid leukaemia – case report
PublicationIn patients with haematological malignancies, the bowel remains the main source of Escherichia coli bloodstream infections. We present the clinical example of recurrent bowel-blood translocations of E. coli with the unique virulence characteristics in a 55-year-old male with the diagnosis of acute myeloid leukaemia. The virulent factors profile of examined strains confirmed that the co-existence of genes papC, sfa, usp and cnf1,...
Year 2016
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A new assay for the simultaneous identification and differentiation of Klebsiella oxytoca strains.
PublicationKlebsiella oxytoca is the second most frequently identified species of Klebsiella isolated from hospitalized patients. Klebsiella spp. is difficult to identify using conventional methods and is often misclassified in clinical microbiology laboratories. K. oxytoca is responsible for an increasing number of multi-resistant infections in hospitals because of insufficient detection and identification. In this study, we propose a new...
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Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms
PublicationA significant number of DNA-based techniques has been introduced into the field of microorganisms’ characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before...
Year 2015
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A new assay for simultaneous identification and differentiation of Klebsiella oxytoca strains
PublicationBacterial strain typing, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics.
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Characterisation of Escherichia coli isolates from the blood of haematological adult patients with bacteraemia: translocation from gut to blood requires the cooperation of multiple virulence factors
PublicationThe aim of the study was to investigate whether there are unique pathotypes of Escherichia coli capable of transmission from the gastrointestinal tract to the vascular bed. The study included E. coli strains isolated from clinical materials collected from 115 patients suffering from haematologic malignancies diagnosed with bacteraemia. The genotyping techniques established that 89 E. coli isolates from the blood had the same genotype...
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Increased concentration of Taq DNA polymerase as a solution for GC-rich templates from clinical and environmental samples
PublicationDNA polymerase is an enzyme which plays crucial role in replication and DNA repair. It found application in PCR (polymerase chain reaction) where catalyses process of in vitro DNA synthesis. To meet the demands posed by mod- ern diagnostic, molecular biology or genetic engineering it is necessary to improve DNA polymerases to obtain new or better features useful in these fields. So far implemented modifications in majority are...
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Novel application of terminal restriction fragments length polymorphism method (t-RFLP) for identification of six clinically important Candida sp.
PublicationTerminal restriction fragments length polymorphism method (t-RFLP) is a modified classical RFLP method. The main difference is based on carrying out PCR using a fluorescently labeled primer or primers. The result of such modification is that after digestion of amplicons only terminal fragments of PCR product (containing fluorescent dye) are visualized. T-RFLP method has found application in environmental microbiology and epidemiology....
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Taq DNA polymerase fused with DNA binding protein with increased resistance to inhibitors from clinical samples
PublicationNowadays PCR method is commonly used in molecular diagnostic. However, in many cases PCR is limited, by the presence of inhibitory substances in biological, soil or food samples Efficiency and fidelity of amplification is strongly connected with DNA polymerase and reaction conditions. To meet the requirements of modern diagnostic methods it is essential to seeking for new DNA polymerases with better properties useful in these field....
Year 2014
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A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model
PublicationWe have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the...
Year 2013
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A subset of two adherence systems, acute pro-inflammatory pap genes and invasion coding dra, fim, or sfa, increases the risk of Escherichia coli translocation to the bloodstream
PublicationAn analysis of the phylogenetic distribution and virulence genes of Escherichia coli isolates which predispose this bacteria to translocate from the urinary tract to the bloodstream is presented. One-dimensional analysis indicated that the occurrence of P fimbriae and α-hemolysin coding genes is more frequent among the E. coli which cause bacteremia. However, a two-dimensional analysis revealed that a combination of genes coding...
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Leukemia and risk of recurrent Escherichia coli bacteremia: genotyping implicates E. coli translocation from the colon to the bloodstream.
PublicationIn patients with leukemia, the portal(s) and reasons for the persistence of an Escherichia coli recurrent bacteremia remain unclear. Adult Hematology Clinic (AHC) databases at the State Clinical Hospital in Gdańsk were reviewed to evaluate the frequency of E. coli bacteremia between 2002 and 2005. Blood and bowel E. coli strains were obtained and the genetic relatedness of the strains was analyzed. The rate of E. coli bacteremia...
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Ligation Mediated PCR methods, their possibilities and limitations
PublicationThe application of molecular diagnostics for bacterial strain typing or identifying bacteria at the strain level is of particular importance at present. However, among the many genotyping methods that are currently available, no single one is universally ideal. In this article, we thus review the Ligation Mediated PCR group methods as prospects from which to choose. We discuss different strategies for selecting the amplified PCR...
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New PCR test for detection of Candida glabrata based on the molecular target chosen by the RAPD technique
PublicationRapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. #e paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted...
Year 2011
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DDLMS PCR double digestion Ligation Mediated Suppression PCR - a new technique for bacterial specific differentiation
PublicationA new diagnostic kit for K. oxytoca specific differentiation based on ddLMS PCR (ang. double digest ligation Mediated PCR) technique is shown. As a species-specific DNA fragment pehX gene, encoding the enzyme polygalactouronase, was chosen. The genome sequence of K. oxytoca is digested with two endonucleases: AclI and BclI which cut DNA before and after pehX gene. The polymorphic DNA fragments are ligated with AclI-end-specific...
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Fatal sepsis in a pregnant woman with pyelonephritis caused by Escherichia coli bearing Dr and P adhesins: diagnosis based on postmortem strain genotyping
PublicationW pracy został opisany pierwszy śmiertelny przypadek młodej kobiety w ciąży z powodu przewlekłego zapalenia odmiedniczkowego nerek spowodowany przez szczepy E. coli z fimbriami typu Dr + i P. Badania pośmiertne wykazały, że śmierć nastąpiła z powodu posocznicy do której doszło na skutek zakażenia szczepami E. coli Dr + . Odmiedniczkowe zapalenie nerek w ciąży jest związane z wieloma powikłaniami, w tym ograniczeniem wzrostu płodu,...
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Homocitrate synthase gene as a new molecular target in diagnostics of Candida spp. infections
PublicationNew potential molecular target which will be investigated during this research (homocitrate synthase gen) is the innovative target other than ribosomal DNA. This gene encodes enzyme present only in fungi and some Archaea what significantly increases specificity. It also reduces risk of contamination or influence of other factors (reaction with other DNA present in sample derived from bacteria or host cells) what excludes false...
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RecA Proteins from Deinococcus geothermalis and Deinococcus murrayi - Cloning, Purification and Biochemical Characterisation
PublicationEscherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS response and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in gamma-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganism
PublicationTechniques relies on the ligation of appropriates adapters (LM-PCR) as AFLP, PCR MP and ADSRRS are successfully used for epidemiological studies for prokaryotic and eukaryotic microorganisms. In this study we propose a new method, called the LM-PCR/Shifter, based on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base 5' overhangs (Shifter) and the ligation of appropriate oligonucleotide...
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