The 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 in the study. Genetic analysis of E. coli isolates, including phylogenetic grouping and virulence factor characteristics, were done by multiplex PCR. We identified 26 patients with chronic sinusitis, in which 26 E. coli isolates were cultured. The E. coli isolates belonged mainly to pathogenic phylogenetic group B2, and carried multiple virulence genes. Three genes in particular were present in all (100%) of examined isolates, they were (1) marker agn43 gene for forming biofilm, (2) type 1 fimbriae (fimG/H gene) and (3) yersiniabactin receptor (fyuA). Furthermore, a pseudo-phylogenetic tree of virulence genes distribution revealed possible cooperation between agn43, fimG/H, and fyuA in the coding of biofilm formation. Intra-operative-biopsy and culture-based therapy, targeting the isolated E. coli, coincided with long-term resolution of symptoms. This is the first report demonstrating an association between a highly pathogenic E. coli, chronic sinus infection, and resolution of symptoms upon E. coli targeted therapy, a significant finding due to the fact that E. coli has not been considered to be a commensal organism of the oropharynx or sinuses. We postulate that the simultaneous presence of three genes, each coding biofilm formation, may in part account for the chronicity of E. coli sinusitis.

Staphylococci 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. We describe molecular methods used in taxonomy as well as staphylococci characterization. Analysis of the 16S rRNA gene, the gap gene, the hsp60 gene, the dnaJ gene, the tuf gene, the sodA gene, and the ropB gene is used as a tool for identification of Staphylococcus isolates. Moreover, the polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of core genes (16S rRNA, gap, hsp60, dnaJ, tuf) is described, and attention is paid to new molecular methods such as real-time PCR and, among nongenome-based assays, mass spectrometry. We are also presenting methods relying on whole genome analysis, including random amplified polymorphism DNA, pulsed-field gel electrophoresis, ligation-mediated PCR, multilocus sequence typing, and spa typing, that are crucial to determine genotypical relatedness among strains of the same species. In addition, current knowledge of SCC (Staphylococcus Cassette Chromosome) mec classification of methicillin-resistant Staphylococcus aureus isolates and selected SCCmec typing methods are discussed.

In 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, encoding virulence factors, predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed. The close cooperation between haematologists and microbiologists is essential to improve the outcome of patients colonised with highly pathogenic strains.

The Polish Arctowski Station is situated in the maritime Antarctic on the western shore of Admiralty Bay and encompasses terrestrial habitats which are not permanently covered by ice, in contrast to more than 90% of the island's surface area. Over the past several decades, studies exploring the soils of those habitats have revealed a considerable diversity of bacteria, filamentous fungi, and, to a lesser extent, yeasts; however, characterization of this complex microbiome, especially at the molecular level, is still far from satisfactory. The isolates were assigned to their respective genera and species based on genetic analysis of the D1/D2 and ITS1-5.8S-ITS2 regions of rDNA. In the studied soil samples, the most abundant microorganisms belonged to the genera Cryptococcus, Rhodotorula, and Debaryomyces. Physiological and biochemical analysis of Cryptococcus gilvescens (pro tempore Goffeauzyma gilvescens) and Rhodotorula mucilaginosa showed only a limited level of intraspecies diversity. Cellular DNA content and karyotypes were determined using flow cytometry and pulsed-field gel electrophoresis for several selected strains. For the first time, genome size and electrophoretic karyotypes were investigated in C. gilvescens (pro tem G. gilvescens), Cryptococcus saitoi (pro tem Naganishia globosa), Cryptococcus gastricus (pro tem Goffeauzyma gastrica), and Cryptococcus albidus (pro tem Naganishia albida). In addition, plate tests showed Antarctic yeasts to be a potential source of biotechnologically important enzymes. This study in biodiversity, presenting physiological and molecular characterization of psychrotolerant yeast strains isolated from the soils of western Admiralty Bay, contributes to a better understanding of the microbial ecology of this unique ecosystem.

Background: Toxoplasma gondii is an important cause of reproductive loss in sheep and has a large economic impact, worldwide, because of infections in humans and animals. The immune response in sheep was evaluated, following administration with four different combinations of recombinant proteins derived from T. gondii.Methods: 25 three-year-old non-pregnant Coopworth ewes were vaccinated twice by intramuscular injection with either the recombinant surface antigen 1 (SAG1), 2 (SAG2) or excretory/secretory antigen (GRA1) each individually conjugated to non-toxic muramyl dipeptide (MDP). Blood samples were collected from all animals prior to the first injection and once per week until five weeks after the second injection. Immunoglobulins (Ig) G1 and IgG2 level were measured using an ELISA test. IFN-gamma were estimated using the commercially available bovine IFN-gamma test kit (Bovigam, Prionics AG).Results: Administration of the recombinant GRA1 enhanced both IFN-gamma production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and GRA1-specific IgG2 antibody level present in serum. Unlike GRAI SAG1 did not stimulate IFN-gamma production in the same test system.Conclusion: These results indicate the potential of recombinant GRA1, as a vaccine candidate to protect sheep against T. gondii infection. (C) 2017 Elsevier B.V. All rights reserved.

PCR 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 used, is shown. In addition, the reasons that might justify the need for modification of DNA polymerases, type of modifications, and links between modified DNA polymerases and PCR efficiency are described.

β-d-Galactosidases have been studied extensively in terms of their application to a variety of industrial technologies. To date, considerable research efforts have been devoted to characterization of new cold-active β-D-galactosidases which were isolated directly from selected species of bacteria and yeasts, as well as with the use of metagenomic approaches. This chapter will provide a review of current research towards cold-active β-D-galactosidases, focusing on the evaluation of the biochemical properties of this group of enzymes and their biotechnological potential in terms of their application to the enzymatic hydrolysis of lactose in milk and milk-based foodstuffs for people with lactose intolerance; the enzymatic synthesis of galactooligosaccharides and heterooligosaccharides (e.g., lactulose); the enzymatic synthesis of alkyl glycosides and nonionic surfactants, which can be used in chemical and pharmaceutical industries; and the enzymatic synthesis of functional food ingredients such as d-tagatose, which is a low-calorie, tooth-friendly sweetener that can be used in confectionery, beverages and dietary products.

This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

A 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 DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.

The crystal structure of a novel dimeric β-D-galactosidase from Paracoccus sp. 32d (ParβDG) was solved in space group P212121 at a resolution of 2.4 Å by molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

Cold active lipolytic enzymes are promising to replace the conventional enzymes processes of biotechnological industries. One of the most important feature of the cold-active lipases and esterases is that they offer economic benefits through energy saving. In general, they exhibit high activity at low temperatures and low thermostability at moderate temperatures. Lipolytic enzyme EstS9 was isolated from Pseudomonas sp. S9. A multiple sequence alignment identified EstS9 as belonging to clade II of the GDSL esterase family. The nucleotide sequence of the estS9 gene of Pseudomonas sp. S9 consists of 1,911 bp. The gene encodes for a protein of 636 amino acid residues with a molecular mass of ∼68.0 kDa. The enzyme is tolerant to alkaline pH and effective at medium to low temperatures (40–25 °C). In our previous study [1], the gene estS9 was expressed using pBAD system in E. coli TOP10 cells as His-tagged fusion protein. Like many other recombinant proteins, EstS9 was produced in E. coli in inclusion bodies. For these reason, there is interest in obtaining the extracellular active esterase which can be used for number of industrial application such as additives for laundry detergents. Therefore, during this study we constructed several recombinant P. pastoris designed for this purpose. Currently, these strains are tested for their ability to produce to the culture medium the active and soluble EstS9 esterase.

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

Sodium hypochlorite (NaClO) is the most commonly used disinfectant in pool treatment system. Outdoor pools usually suffer from the strong sunlight irradiation which degrades the free chlorine rapidly. In addition, more pools start to adopt the recirculation of swimming pool water, which intensifies the disinfection by-product (DBP) accumulation issue. Given these potential drawbacks of using NaClO in the tropical environment, two alternative organic-based disinfectants, trichloroisocyanuric acid (TCCA, C3Cl3N3O3) and bromochlorodimethylhydantoin (BCDMH, C5H6BrClN2O2), were investigated and compared to NaClO in terms of their self-degradation and the formation of DBPs, including trihalomethanes (THMs) and haloacetic acids (HAAs), under simulated tropical climate conditions. The result reveals that halogen stabilizer, TCCA, had the advantages of slower free chlorine degradation and lower DBP concentration compared to NaClO, which makes it a good alternative disinfectant. BCDMH was not recommended mainly due to the highly reactive disinfecting ingredient, hypobromous acid (HBrO), which fails to sustain the continuous disinfection requirement. Total disinfectant dosage was the main factor that affects residual chlorine/bromine and THM/HAA formation regardless of different disinfectant dosing methods, e.g. shock dosing (one-time spiking) in the beginning, and continuous dosing during the whole experimental period. Two-stage second-order-kinetic-based models demonstrate a good correlation between the measured and predicted data for chlorine decay (R2 ≥ 0.95), THM (R2 ≥ 0.99) and HAA (R2 ≥ 0.83) formation. Higher temperature was found to enhance the DBP formation due to the temperature dependence of reaction rates. Thus, temperature control of pools, especially for those preferring higher temperatures (e.g. hydrotherapy and spa), should take both bather comfort and DBP formation potential into consideration. It is also observed that chlorine competition existed between different precursors from natural organic matters (NOM) in filling water and body fluid analogue (BFA). Among the composition of BFA, uric acid, citric acid and hippuric acid were found to be the main precursors for HAA formation.

The aim of the study was to evaluate the usefulness of 3 chimeric Toxoplasma gondii antigens, P35-MAG1, MIC1-ROP1 and MAG1-ROP1, in the serodiagnosis of an acute toxoplasmosis in humans. Proteins were produced as fusion proteins containing His tags ends and then further purified by metal affinity chromatography. Their application for the diagnosis of recently acquired T. gondii infection was tested in IgG and IgM enzyme-linked immunosorbent assays (ELISAs). At 100%, 77.3%, and 86.4%, respectively, the reactivity of the IgG ELISA using P35-MAG1, MIC1-ROP1, and MAG1-ROP1 for sera from patients where acute toxoplasmosis was suspected was significantly higher than for the samples from people with a chronic infection, at 26.2%, 36.1%, and 32.8%, respectively. Moreover, P35-MAG1, MIC1-ROP1, and MAG1-ROP1 detected IgM antibodies with a reactivity at 81.8%, 72.7%, and 59.1%, respectively. The results presented in the article show that, particularly, P35-MAG1 may be useful in the preliminary detection of recent T. gondii infection.

INTRODUCTION:Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking. METHODS:Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice. RESULTS:Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii. CONCLUSION:In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further vaccine development against T. gondii infection.

BACKGROUND:SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis.RESULTS:This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m)) is 100.2 °C as shown by differential scanning calorimetry (DSC) analysis.CONCLUSION:NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly used TLA.

The 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 as the E. coli from the patient's bowel. The presence of 21 genes encoding virulence factors typical of various E. coli pathotypes and their relationship with the phylogenetic group was established. One-dimensional analysis showed that the focG gene occurred more frequently in the control bowel group, while the ampicillin-resistant afa/dr E. coli were associated with bacteraemia. Blood isolates with the highest occurrence of virulence factors belonged to pathogenic group B2 and non-pathogenic group A. The co-occurrence of multiple genes encoding papC, sfa, usp and cnf1 virulence factors probably predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed in the group of patients with haematologic malignancies. Based on clustering analysis, dominance of the most virulent strains assigned to the cluster with seven virulence factors encoded by the following genes, papC, sfaD/E, cnf1, usp, agn43, hlyA and iutA, was found. The obtained results enforce the previously proposed concept of bowel-blood translocation and further expand our hypothesis by defining the unique virulence characteristics of E. coli isolates, which predispose them to bowel colonisation or translocation and bacteraemia in this group of patients.