adhesiomeR: a tool for Escherichia coli adhesin classification and analysis.

Adhesins are crucial factors in the virulence of bacterial pathogens such as Escherichia coli. However, to date no resources have been dedicated to the detailed analysis of E. coli adhesins. Here, we provide adhesiomeR software that enables characterization of the complete adhesin repertoire, termed the adhesiome. AdhesiomeR incorporates the most comprehensive database of E. coli adhesins and facilitates an extensive analysis of adhesiome. We demonstrate that adhesiomeR achieves 98% accuracy when compared with experimental analyses. Based on analysis of 15,000 E. coli genomes, we define novel adhesiome profiles and clusters, providing a nomenclature for a unified comparison of E. coli adhesiomes.

Correction: Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins.

[This corrects the article DOI: 10.1371/journal.ppat.1010118.].

Characterization of Salmonella enterica Biofilms and Antibiofilm Effect of Carvacrol and 2-Aminobenzimidazole.

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica, namely Salmonella Typhimurium (n = 23), Salmonella Infantis (n = 28), and Salmonella Heidelberg (n = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB). Seven of the tested isolates exhibited moderate biofilm formation in diluted TSB, whereas two showed such formation in RVS. In addition, the Congo red agar assay revealed curli and cellulose production in seven isolates. Fourteen specific biofilm-associated genes were analyzed identifying sdiA and seqA to be the most prevalent (100%), and glyA the least prevalent (69.5%). The prevalence of the genes bcsA and csgA was significantly lower in moderate and weak biofilm formers, respectively, as compared with nonbiofilm formers in RVS peptone broth. Furthermore, the compounds carvacrol and 2-aminobenzimidazole (2-ABI) effectively inhibited biofilm formation by Salmonella serovars in RVS peptone and TSB media, respectively. Whereas the antibiofilm activity of 2-ABI against Salmonella has not been reported previously, we determined its most effective concentration at 1.5 mM among tested antibiofilm treatments. These findings indicate that Salmonella strains prevalent in poultry farms have the potential to form biofilms, and the tested compounds should be further explored as supportive or alternative antimicrobials.

Human glycoprotein-2 expressed in Brunner glands - A putative autoimmune target and link between Crohn's and coeliac disease.

Glycoprotein 2 (GP2) is an autoantigen in Crohn's (CD) and coeliac disease (CeD). We assessed GP2-isoform (GP21-4)-expression in intestinal biopsies of paediatric patients with CD, CeD, ulcerative colitis (UC), and healthy children (HC). Transcription of GP21-4 was elevated in proximal small intestine in CeD and CD patients (only GP22/4) compared to jejunum (CeD/CD) and large bowel (CD). CeD patients demonstrated higher duodenal GP22/4-mRNA levels compared to HC/UC patients whereas CD patients showed higher GP24-mRNA levels compared to UC patients. Duodenal synthesis of only small GP2 isoforms (GP23/4) was demonstrated in epithelial cells in patients/HC and in Brunner glands (also large isoforms) with a more frequent apical location in CD/CeD patients. All four GP2 isoforms interacted with gliadin and phosphopeptidomannan. Gliadin digestion improved binding to GP2 isoforms. GP21-4 binding to CeD/CD-related antigens, elevated duodenal GP21-4-mRNA transcription, and GP2-protein secretion in Brunner glands of CeD/CD patients suggest an autoimmune CeD/CD link.

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins.

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity in targeting phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or ß2-glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by-among other factors-viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL with prothrombin (PT). The strength of the antibody response against SARS-CoV-2 was further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity, and aPL against PT in patients with SARS-CoV-2.

The role of AJB35136 and fdtA genes in biofilm formation by avian pathogenic Escherichia coli.

BACKGROUND: Infections caused by avian pathogenic Escherichia coli (APEC) result in significant economic losses in poultry industry. APEC strains are known to form biofilms in various conditions allowing them to thrive even under harsh and nutrient-deficient conditions on different surfaces, and this ability enables them to evade chemical and biological eradication methods. Despite knowing the whole genome sequences of various APEC isolates, little has been reported regarding their biofilm-associated genes. A random transposon mutant library of the wild-type APEC IMT 5155 comprising 1,300 mutants was analyzed for biofilm formation under nutrient deprived conditions using Videoscan technology coupled with fluorescence microscopy. Seven transposon mutants were found to have reproducibly and significantly altered biofilm formation and their mutated genes were identified by arbitrary PCR and DNA sequencing. The intact genes were acquired from the wild-type strain, cloned in pACYC177 plasmid and transformed into the respective altered biofilm forming transposon mutants, and the biofilm formation was checked in comparison to the wild type and mutant strains under the same conditions. RESULTS: In this study, we report seven genes i.e., nhaA, fdeC, yjhB, lysU, ecpR, AJB35136 and fdtA of APEC with significant contribution to biofilm formation. Reintroduction of AJB35136 and fdtA, reversed the altered phenotype proving that a significant role being played by these two O-antigen related genes in APEC biofilm formation. Presence of these seven genes across nonpathogenic E. coli and APEC genomes was also analyzed showing that they are more prevalent in the latter. CONCLUSIONS: The study has elucidated the role of these genes in APEC biofilm formation and compared them to adhesion expanding the knowledge and understanding of the economically significant pathogens.

Over-Expression of LEDGF/p75 in HEp-2 Cells Enhances Autoimmune IgG Response in Patients with Benign Prostatic Hyperplasia-A Novel Diagnostic Approach with Therapeutic Consequence?

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) is an autoantigen over-expressed in solid tumors and acts as a stress-related transcriptional co-activator. Participation of autoimmune responses in the pathophysiology of benign prostatic hyperplasia (PBH) and a corresponding immunosuppressive therapy by TNFalpha antagonists has been recently suggested. Thus, autoAb testing could aid in the diagnosis of BPH patients profiting from such therapy. We generated CRISPR/Cas9 modified HEp-2 LEDGF knock-out (KO) and HEp-2 LEDGF/p75 over-expressing (OE) cells and examined IgG autoantibody reactivity to LEDGF/p75 in patients with prostate cancer (PCa, n = 89), bladder cancer (BCa, n = 116), benign prostatic hyperplasia (BPH, n = 103), and blood donors (BD, n = 60) by indirect immunofluorescence assay (IFA). Surprisingly, we could not detect elevated binding of autoAbs against LEDGF/p75 in cancer patients, but autoAb reactivity to LEDGF/p75 OE cells in about 50% of patients with BPH was unexpectedly significantly increased. Furthermore, a line immunoassay enabling the detection of 18 different autoAbs revealed a significantly increased occurrence of anti-dsDNA autoAbs in 34% of BPH patients in contrast to tumor patients and BD. This finding was confirmed by anti-mitochondrial (mDNA) autoAb detection with the Crithidia luciliae immunofluorescence test, which also showed a significantly higher prevalence (34%) of anti-mDNA autoAbs in BPH. In summary, our study provided further evidence for the occurrence of autoimmune responses in BPH. Furthermore, LEDGF/p75 over-expression renders HEp-2 cells more autoantigenic and an ideal target for autoAb analysis in BPH with a potential therapy consequence.

Adhesion of Enteropathogenic, Enterotoxigenic, and Commensal Escherichia coli to the Major Zymogen Granule Membrane Glycoprotein 2.

Pathogenic bacteria, such as enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC), cause diarrhea in mammals. In particular, E. coli colonizes and infects the gastrointestinal tract via type 1 fimbriae (T1F). Here, the major zymogen granule membrane glycoprotein 2 (GP2) acts as a host cell receptor. GP2 is also secreted by the pancreas and various mucous glands, interacting with luminal type 1 fimbriae-positive E. coli. It is unknown whether GP2 isoforms demonstrate specific E. coli pathotype binding. In this study, we investigated interactions of human, porcine, and bovine EPEC and ETEC, as well as commensal E. coli isolates with human, porcine, and bovine GP2. We first defined pathotype- and host-associated FimH variants. Second, we could prove that GP2 isoforms bound to FimH variants to various degrees. However, the GP2-FimH interactions did not seem to be influenced by the host specificity of E. coli. In contrast, soluble GP2 affected ETEC infection and phagocytosis rates of macrophages. Preincubation of the ETEC pathotype with GP2 reduced the infection of cell lines. Furthermore, preincubation of E. coli with GP2 improved the phagocytosis rate of macrophages. Our findings suggest that GP2 plays a role in the defense against E. coli infection and in the corresponding host immune response. IMPORTANCE Infection by pathogenic bacteria, such as certain Escherichia coli pathotypes, results in diarrhea in mammals. Pathogens, including zoonotic agents, can infect different hosts or show host specificity. There are Escherichia coli strains which are frequently transmitted between humans and animals, whereas other Escherichia coli strains tend to colonize only one host. This host specificity is still not fully understood. We show that glycoprotein 2 is a selective receptor for particular Escherichia coli strains or variants of the adhesin FimH but not a selector for a species-specific Escherichia coli group. We demonstrate that GP2 is involved in the regulation of colonization and infection and thus represents a molecule of interest for the prevention or treatment of disease.

Identification of Natural Mutations Responsible for Altered Infection Phenotypes of Salmonella enterica Clinical Isolates by Using Cell Line Infection Screens.

The initial steps of Salmonella pathogenesis involve adhesion to and invasion into host epithelial cells. While well-studied for Salmonella enterica serovar Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium, and Enteritidis for adhesion to and invasion into intestinal epithelial cell lines of human, porcine, and chicken origins. Thirty isolates with altered infectivity were used for genomic analyses, and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation and cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion to and invasion into cells has not previously been investigated. The genes dksA (encoding a stringent response regulator) and sanA (encoding a vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in adhesion to and invasion into host cells. Furthermore, transcriptomic analyses were performed for S Enteritidis and S Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of type 3 secretion system expression levels in the determination of an isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion to and invasion into the epithelial cells.IMPORTANCESalmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Its adhesion to and invasion into intestinal epithelial cells represent one of the first and key steps in the pathogenesis of salmonellosis. Still, around 35 to 40% of bacterial genes have no experimentally validated function, and their contribution to bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore, the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection; therefore, the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative, as the prevalence of antibiotic-resistant bacteria is increasing very rapidly.

Fluorescence-encoded poly(methyl metharcylate) nanoparticles for a lateral flow assay detecting IgM autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) belongs to the most often occurring autoimmune diseases in the world. For serological diagnosis, IgM auto-antibodies directed against the Fc portion of IgG referred to as rheumatoid factor are used as biomarkers. The autoantibody detection is usually done by ELISA. Such assays are reliable but are not suitable for point-of-care testing in contrast to lateral flow assays. Here, we report the development of a lateral flow assay based on carboxylated fluorescence-encoded poly(methyl methacrylate) nanoparticles. Poly(methyl methacrylate) is a non-toxic plastic with an excellent biocompatibility and high optical transparency which promises especially high sensitive fluorescence detection thereby leading to very sensitive assays. We could detect a positive signal in samples with a nephelometric reading down to 0.4 U/mL. By analyzing 30 sera of patients with a RA diagnosis and 34 sera of healthy test subjects we could confirm positive ELISA results in 72% of all cases and negative ELISA results in 97% of all cases.

Chitinase 3-like 1 is not a target antigen in patients with multiple sclerosis.

Autoimmunity to chitinase 3-like 1 (CHI3L1) has recently been reported in hepatic and bowel inflammatory conditions. Considering that CHI3L1 plays a role as prognostic biomarker in multiple sclerosis (MS), here we investigated CHI3L1 as potential autoantigenic target in the disease. We determined serum CHI3L1 autoantibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 60 untreated MS patients with different clinical forms of the disease and 20 healthy controls (HC). IgG levels to CHI3L1 were similar between patients with relapsing-remitting MS, primary and secondary progressive MS, and HC. These findings do not support a role for CHI3L1 autoantibodies in MS pathogenesis.

Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections.

Infections caused by carbapenem-resistant Pseudomonas aeruginosa are life-threatening due to its synergistic resistance mechanisms resulting in the ineffectiveness of the used antimicrobials. This study aimed to characterize P. aeruginosa isolates for antimicrobial susceptibility, biofilm formation virulence genes, and molecular mechanisms responsible for resistance against various antimicrobials. Out of 700 samples, 91 isolates were confirmed as P. aeruginosa which were further classified into 19 non-multidrug-resistant (non-MDR), 7 multidrug-resistant (MDR), 19 extensively drug-resistant (XDR), and 8 pan drug-resistant (PDR) pulsotypes based on standard Kirby Bauer disc diffusion test and pulse field gel electrophoresis. In M9 minimal media, strong biofilms were formed by the XDR and PDR pulsotypes as compared to the non-MDR pulsotypes. The virulence genes, responsible for the worsening of wounds including LasB, plcH, toxA, and exoU, were detected among all MDR, XDR, and PDR pulsotypes. Carbapenemase activity was phenotypically detected in 45% pulsotypes and the responsible genes were found as blaGES (100%), blaVIM (58%), blaIMP (4%), and blaNDM (4%). Real-time polymerase chain reaction showed the concomitant use of multiple mechanisms such as oprD under-expression, enhanced efflux pump activity, and ampC overexpression in the resistant isolates. Polymyxin is found as the only class left with more than 80% susceptibility among the isolates which is an alarming situation suggesting appropriate measures to be taken including alternative therapies. KEY POINTS: • Multidrug-resistant P. aeruginosa isolates formed stronger biofilms in minimal media. • Only polymyxin antimicrobial was found effective against MDR P. aeruginosa isolates. • Under-expression of oprD and overexpression of ampC were found in resistant isolates.

LEDGF/p75 Is Required for an Efficient DNA Damage Response.

Lens epithelium-derived growth factor splice variant of 75 kDa (LEDGF/p75) plays an important role in cancer, but its DNA-damage repair (DDR)-related implications are still not completely understood. Different LEDGF model cell lines were generated: a complete knock-out of LEDGF (KO) and re-expression of LEDGF/p75 or LEDGF/p52 using CRISPR/Cas9 technology. Their proliferation and migration capacity as well as their chemosensitivity were determined, which was followed by investigation of the DDR signaling pathways by Western blot and immunofluorescence. LEDGF-deficient cells exhibited a decreased proliferation and migration as well as an increased sensitivity toward etoposide. Moreover, LEDGF-depleted cells showed a significant reduction in the recruitment of downstream DDR-related proteins such as replication protein A 32 kDa subunit (RPA32) after exposure to etoposide. The re-expression of LEDGF/p75 rescued all knock-out effects. Surprisingly, untreated LEDGF KO cells showed an increased amount of DNA fragmentation combined with an increased formation of γH2AX and BRCA1. In contrast, the protein levels of ubiquitin-conjugating enzyme UBC13 and nuclear proteasome activator PA28γ were substantially reduced upon LEDGF KO. This study provides for the first time an insight that LEDGF is not only involved in the recruitment of CtIP but has also an effect on the ubiquitin-dependent regulation of DDR signaling molecules and highlights the role of LEDGF/p75 in homology-directed DNA repair.

Novel Avian Pathogenic Escherichia coli Genes Responsible for Adhesion to Chicken and Human Cell Lines.

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen of commercial poultry contributing to extensive economic losses and contamination of the food chain. One of the initial steps in bacterial infection and successful colonization of the host is adhesion to the host cells. A random transposon mutant library (n = 1,300) of APEC IMT 5155 was screened phenotypically for adhesion to chicken (CHIC-8E11) and human (LoVo) intestinal epithelial cell lines. The detection and quantification of adherent bacteria were performed by a modified APEC-specific antibody staining assay using fluorescence microscopy coupled to automated VideoScan technology. Eleven mutants were found to have significantly altered adhesion to the cell lines examined. Mutated genes in these 11 "adhesion-altered mutants" were identified by arbitrary PCR and DNA sequencing. The genes were amplified from wild-type APEC IMT 5155, cloned, and transformed into the respective adhesion-altered mutants, and complementation was determined in adhesion assays. Here, we report contributions of the fdtA, rluD, yjhB, ecpR, and fdeC genes of APEC in adhesion to chicken and human intestinal cell lines. Identification of the roles of these genes in APEC pathogenesis will contribute to prevention and control of APEC infections.IMPORTANCE Avian pathogenic E. coli is not only pathogenic for commercial poultry but can also cause foodborne infections in humans utilizing the same attachment and virulence mechanisms. Our aim was to identify genes of avian pathogenic E. coli involved in adhesion to chicken and human cells in order to understand the colonization and pathogenesis of these bacteria. In contrast to the recent studies based on genotypic and bioinformatics data, we have used a combination of phenotypic and genotypic approaches for identification of novel genes contributing to adhesion in chicken and human cell lines. Identification of adhesion factors remains important, as antibodies elicited against such factors have shown potential to block colonization and ultimately prevent disease as prophylactic vaccines. Therefore, the data will augment the understanding of disease pathogenesis and ultimately in designing strategies against the infections.

Streptavidin Homologues for Applications on Solid Surfaces at High Temperatures.

One of the most commonly used bonds between two biomolecules is the bond between biotin and streptavidin (SA) or streptavidin homologues (SAHs). A high dissociation constant and the consequent high-temperature stability even allows for its use in nucleic acid detection under polymerase chain reaction (PCR) conditions. There are a number of SAHs available, and for assay design, it is of great interest to determine as to which SAH will perform the best under assay conditions. Although there are numerous single studies on the characterization of SAHs in solution or selected solid phases, there is no systematic study comparing different SAHs for biomolecule-binding, hybridization, and PCR assays on solid phases. We compared streptavidin, core streptavidin, traptavidin, core traptavidin, neutravidin, and monomeric streptavidin on the surface of microbeads (10-15 µm in diameter) and designed multiplex microbead-based experiments and analyzed simultaneously the binding of biotinylated oligonucleotides and the hybridization of oligonucleotides to complementary capture probes. We also bound comparably large DNA origamis to capture probes on the microbead surface. We used a real-time fluorescence microscopy imaging platform, with which it is possible to subject samples to a programmable time and temperature profile and to record binding processes on the microbead surface depending on the time and temperature. With the exception of core traptavidin and monomeric streptavidin, all other SA/SAHs were suitable for our investigations. We found hybridization efficiencies close to 100% for streptavidin, core streptavidin, traptavidin, and neutravidin. These could all be considered equally suitable for hybridization, PCR applications, and melting point analysis. The SA/SAH-biotin bond was temperature-sensitive when the oligonucleotide was mono-biotinylated, with traptavidin being the most stable followed by streptavidin and neutravidin. Mono-biotinylated oligonucleotides can be used in experiments with temperatures up to 70 °C. When oligonucleotides were bis-biotinylated, all SA/SAH-biotin bonds had similar temperature stability under PCR conditions, even if they comprised a streptavidin variant with slower biotin dissociation and increased mechanostability.

The loss of tolerance to CHI3L1 - A putative role in inflammatory bowel disease?

The incidence of inflammatory bowel disease (IBD) is steadily increasing. IBD is characterized by chronic inflammation of the gastrointestinal tract and is divided into the two main entities Crohn's disease (CD) and ulcerative colitis (UC). Genetic predispositions, environmental factors and a dysregulated immune response are known to be involved at the beginning of IBD. However, their etiopathogenesis is not yet fully understood. Over the last ten years, there has been increasing evidence of the involvement of the member of the 18-glycosylhydrolase family chitinase-3-like protein 1 (CHI3L1) in IBD. CHI3L1 is associated with various diseases such as cancer and chronic inflammatory diseases including rheumatoid arthritis or IBD as well as neurological diseases where it can act as a chemoattractant, mitogen or growth factor. This review will focus on the role of autoimmunity to CHI3L1 in IBD in the context of its expression in inflamed colonic epithelia and interaction with intestinal microbiota. Further, it will provide insights into the interaction of CHI3L1 with different mechanisms of the innate and adaptive immune response in IBD.

Spatial Separation of Microbeads into Detection Levels by a Bioorthogonal Porous Hydrogel for Size-Selective Analysis and Increased Multiplexity.

Multiplex detection techniques are emerging within the fields of life science research and medical diagnostics where it is mandatory to analyze a great number of molecules. The detection techniques need to be highly efficient but often involve complicated and expensive fabrication procedures. Here, we present the immobilization and geometric separation of fluorescence-labeled microbeads for a multiplex detection in k levels. A compound of differently sized target molecules (DNA, proteins) is channeled into the respective detection levels by making use of a hydrogel as a size selective filter. The immobilized microbeads (10-20 µm) are considerably larger than the pores of the hydrogel network and therefore stay fixed at the well bottom and in higher elevations, respectively. Small biomolecules can diffuse through the pores of the network, whereas medium-sized biomolecules pass slower and large molecules will be excluded. Besides filtering, this method discriminates the used microbeads into k levels and thereby introduces a geometric multiplexity. Additionally, the exclusion of large entities enables the simultaneous detection of two target molecules, which exhibit the same affinity interaction. The hydrogel is formed through the combination of two macromonomers. One component is a homobifunctional polyethylene glycol linker, carrying a strained alkyne (PEG-BCN) and the second component is the azide-functionalized dendritic polyglycerol (dPG-N3). They react via the bioorthogonal strain-promoted azide alkyne cycloaddition (SPAAC). The hydrogel creates a solution-like environment for the diffusion of the investigated biomolecules all the while providing a stable, bioinert, and surface bound network.

Simultaneous detection and quantification of DNA and protein biomarkers in spectrum of cardiovascular diseases in a microfluidic microbead chip.

The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11-15 µm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization. Graphical abstract.

Comparative Analysis of Draft Genome Sequence of Rhodococcus sp. Eu-32 with Other Rhodococcus Species for Its Taxonomic Status and Sulfur Metabolism Potential.

Rhodococcus sp. Eu-32 has shown an extended novel dibenzothiophene desulfurization sulfur-specific 4S pathway and could remove significant amounts of organic sulfur from coal. Here, we present the draft genome sequence of Eu-32 with a genome size of approximately 5.61 Mb, containing 5065 protein coding sequences with a G+C content of 65.1%. The Rhodococcus sp. Eu-32 showed ~ 99% identity at the 16S rRNA gene sequence level while < 34% digital DNA-DNA hybridization and < 81% average nucleotide identity values with the genome sequence of most closely related known Rhodococcus species, suggesting that it is taxonomically different from the already reported Rhodococcus species. Among the annotated genes, 90 are involved in the metabolism of sulfur. Comparative genome analysis suggests many commonalities in sulfur metabolism gene sets that may have evolved due to many factors including ecological pressures. Our study and the genome sequence data will be available for further research and will provide insights into potential biotechnological and industrial applications of this bacterium.

Genotypic and Phenotypic Characteristics Associated with Biofilm Formation by Human Clinical Escherichia coli Isolates of Different Pathotypes.

Bacterial biofilm formation is a widespread phenomenon and a complex process requiring a set of genes facilitating the initial adhesion, maturation, and production of the extracellular polymeric matrix and subsequent dispersal of bacteria. Most studies on Escherichia coli biofilm formation have investigated nonpathogenic E. coli K-12 strains. Due to the extensive focus on laboratory strains in most studies, there is poor information regarding biofilm formation by pathogenic E. coli isolates. In this study, we genotypically and phenotypically characterized 187 human clinical E. coli isolates representing various pathotypes (e.g., uropathogenic, enteropathogenic, and enteroaggregative E. coli). We investigated the presence of biofilm-associated genes ("genotype") and phenotypically analyzed the isolates for motility and curli and cellulose production ("phenotype"). We developed a new screening method to examine the in vitro biofilm formation ability. In summary, we found a high prevalence of biofilm-associated genes. However, we could not detect a biofilm-associated gene or specific phenotype correlating with the biofilm formation ability. In contrast, we did identify an association of increased biofilm formation with a specific E. coli pathotype. Enteroaggregative E. coli (EAEC) was found to exhibit the highest capacity for biofilm formation. Using our image-based technology for the screening of biofilm formation, we demonstrated the characteristic biofilm formation pattern of EAEC, consisting of thick bacterial aggregates. In summary, our results highlight the fact that biofilm-promoting factors shown to be critical for biofilm formation in nonpathogenic strains do not reflect their impact in clinical isolates and that the ability of biofilm formation is a defined characteristic of EAEC.IMPORTANCE Bacterial biofilms are ubiquitous and consist of sessile bacterial cells surrounded by a self-produced extracellular polymeric matrix. They cause chronic and device-related infections due to their high resistance to antibiotics and the host immune system. In nonpathogenic Escherichia coli, cell surface components playing a pivotal role in biofilm formation are well known. In contrast, there is poor information for their role in biofilm formation of pathogenic isolates. Our study provides insights into the correlation of biofilm-associated genes or specific phenotypes with the biofilm formation ability of commensal and pathogenic E. coli Additionally, we describe a newly developed method enabling qualitative biofilm analysis by automated image analysis, which is beneficial for high-throughput screenings. Our results help to establish a better understanding of E. coli biofilm formation.