Phage Display-Derived Binders Able to Distinguish Listeria monocytogenes from Other Listeria Species

The objective of this study was to produce phage display-derived binders with the ability to distinguish Listeria monocytogenes from other Listeria spp., which may have potential utility to enhance detection of Listeria monocytogenes. To obtain binders with the desired binding specificity a series of surface and solution phage-display biopannings were performed. Initially, three rounds of surface biopanning against gamma-irradiated L. monocytogenes serovar 4b cells were performed followed by an additional surface biopanning round against L. monocytogenes 4b which included prior subtraction biopanning against gamma-irradiated L. innocua cells. In an attempt to further enhance binder specificity for L. monocytogenes 4b two rounds of solution biopanning were performed, both rounds included initial subtraction solution biopanning against L. innocua. Subsequent evaluations were performed on the phage clones by phage binding ELISA. All phage clones tested from the second round of solution biopanning had higher specificity for L. monocytogenes 4b than for L. innocua and three other foodborne pathogens (Salmonella spp., Escherichia coli and Campylobacter jejuni). Further evaluation with five other Listeria spp. revealed that one phage clone in particular, expressing peptide GRIADLPPLKPN, was highly specific for L. monocytogenes with at least 43-fold more binding capability to L. monocytogenes 4b than to any other Listeria sp. This proof-of-principle study demonstrates how a combination of surface, solution and subtractive biopanning was used to maximise binder specificity. L. monocytogenes-specific binders were obtained which could have potential application in novel detection tests for L. monocytogenes, benefiting both the food and medical industries. © 2013 Morton et al.

Development and Validation of a PulseNet Standardized Protocol for Subtyping Isolates of Cronobacter Species

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

METHOD OF DETECTING BIOPRODUCTS USING LOCALIZED SURFACE PLASMON RESONANCE SENSOR OF GOLD NANOPARTICLES

(EN)Disclosed is a method of detecting bioproducts using Localized Surface Plasmon Resonance (LSPR) of gold nanoparticles, which can diagnose bioproducts based on changes in the maximum wavelength occurred by an antigen-antibody reaction after immobilization of the gold nanoparticles onto a glass panel. A sensor using such method exhibits high sensitivity, is low in price, and makes quick diagnosis possible, thereby being applicable to various biological fields associated with environmental contaminants, pathogens and the like, as well as diagnosis of diseases. Further, it provides a technology for manufacturing a sensor having higher sensitivity, low price and quick performance, as compared to conventional methods using SPR.

A Trip from a Tube to a Chip Applied Micro and Nanotechnology in Biotechnology, Veterinary and Life Sciences

More than 200 known diseases are transmitted via foods or food products. In the United States, food-borne diseases are responsible for 76 million cases of illness, 32,500 cases of hospitalisation and 5000 cases of death yearly. The ongoing increase in worldwide trade in livestock, food, and food products in combination with increase in human mobility (business- and leisure travel, emigration etc.) will increase the risk of emergence and spreading of such pathogens. There is therefore an urgent need for development of rapid, efficient and reliable methods for detection and identification of such pathogens.

Microchipfabrication has had a major impact on electronics and is expected to have an equally pronounced effect on life sciences. By combining micro-fluidics with micromechanics, micro-optics, and microelectronics, systems can be realized to perform complete chemical or biochemical analyses. These socalled ’Lab-on-a-Chip’ will completely change the face of laboratories in the future where smaller, fully automated devices will be able to perform assays faster, more accurately, and at a lower cost than equipment of today. A general introduction of food safety and applied micro-nanotechnology in life sciences will be given. In addition, examples of DNA micro arrays, micro fabricated integrated PCR chips and total integrated lab-on-achip systems from different National and EU research projects being carried out at the Laboratory of Applied Micro- Nanotechnology (LAMINATE) group at the National Veterinary Institute (DTU-Vet) Technical University of Denmark and the BioLabchip group at the Department of Micro and Nanotechnology (DTU-Nanotech), Technical University of Denmark (DTU), Ikerlan-IK4 (Spain) and other 16 partners from different European countries will be presented.

Development of M13 bacteriophage-based SPR detection using Salmonella detection as a case study

Surface plasmon resonance (SPR)-based biosensor is a popular platform for real-time monitoring and sensitive detection for a myriad of targets. However, only a few studies have reported the use of bacteriophages as specific binders for SPR-based detection. This study aimed to demonstrate how filamentous M13 bacteriophages expressing 12-mer peptides can be employed in an SPR-based assay, using a Salmonella-specific bacteriophage as a model binder to detect the foodborne bacterium Salmonella. Several important factors (immobilization buffers and methods, and interaction buffers) for a successful bacteriophage-based SPR assay were optimized. As a result, a Salmonella-specific bacteriophage-based SPR assay was achieved, with very low cross reactivity with other non-target foodborne pathogens and detection limits of 8.0 × 10⁷ and 1.3 × 10⁷ CFU/mL for one-time and five-time immobilized sensors, respectively. This proof-of-concept study demonstrates the feasibility of using M13 bacteriophages expressing target-specific peptides as a binder in a rapid and label-free SPR assay for pathogen detection.

The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis.

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.

Development of novel oral formulations prepared via hot melt extrusion for targeted delivery of photosensitizer to the colon

Colon-residing bacteria, such as vancomycin-resistant Enterococcus faecalis and Bacteroides fragilis, can cause a range of serious clinical infections. Photodynamic antimicrobial chemotherapy (PACT) may be a novel treatment option for these multidrug resistant organisms. The aim of this study was to formulate a Eudragit®-based drug delivery system, via hot melt extrusion (HME), for targeting colonic release of photosensitizer. The susceptibility of E. faecalis and B. fragilis to PACT mediated by methylene blue (MB), meso-tetra(N-methyl-4-pyridyl)porphine tetra-tosylate (TMP), or 5-aminolevulinic acid hexyl-ester (h-ALA) was determined, with tetrachlorodecaoxide (TCDO), an oxygen-releasing compound, added in some studies. Results show that, for MB, an average of 30% of the total drug load was released over a 6-h period. For TMP and h-ALA, these values were 50% and 16% respectively. No drug was released in the acidic media. Levels of E. faecalis and B. fragilis were reduced by up to 4.67 and 7.73 logs, respectively, on PACT exposure under anaerobic conditions, with increased kill associated with TCDO. With these formulations, photosensitizer release could potentially be targeted to the colon, and colon-residing pathogens killed by PACT. TCDO could be used in vivo to generate oxygen, which could significantly impact on the success of PACT in the clinic.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

Propionibacterium acnes and Staphylococcus lugdunensis Cause Pyogenic Osteomyelitis in an Intramedullary Nail Model in Rabbits

Propionibacterium acnes and coagulase-negative staphylococci (CoNS) are opportunistic pathogens implicated in prosthetic joint and fracture fixation device-related infections. The purpose of this study was to determine whether P. acnes and the CoNS species Staphylococcus lugdunensis, isolated from an "aseptically failed" prosthetic hip joint and a united intramedullary nail-fixed tibial fracture, respectively, could cause osteomyelitis in an established implant-related osteomyelitis model in rabbits in the absence of wear debris from the implant material. The histological features of P. acnes infection in the in vivo rabbit model were consistent with localized pyogenic osteomyelitis, and a biofilm was present on all explanted intramedullary (IM) nails. The animals displayed no outward signs of infection, such as swelling, lameness, weight loss, or elevated white blood cell count. In contrast, infection with S. lugdunensis resulted in histological features consistent with both pyogenic osteomyelitis and septic arthritis, and all S. lugdunensis-infected animals displayed weight loss and an elevated white blood cell count despite biofilm detection in only two out of six rabbits. The differences in the histological and bacteriological profiles of the two species in this rabbit model of infection are reflective of their different clinical presentations: low-grade infection in the case of P. acnes and acute infection for S. lugdunensis. These results are especially important in light of the growing recognition of chronic P. acnes biofilm infections in prosthetic joint failure and nonunion of fracture fixations, which may be currently reported as "aseptic" failure. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A microbiological survey of a constructed wetland system supplied by dairy 'dirty water' effluent

A constructed wetland at Greenmount College, Co. Antrim, N. Ireland was built in 2004 to study the treatment of ‘dirty water’ effluent from the Greenmount dairy unit. The effluent has a mean BOD5 of c.1000 mg/L and contains milking parlour wash-water and runoff from silage clamps and yard areas lightly contaminated with cattle manure. The nominal water retention time of this wetland is 100 days. The primary purposes of the wetland are to eliminate organic pollution and eutrophication risk from nitrogen and phosphorus compounds. However the wetland should also effectively remove any zoonotic pathogens present in manure and milk. Accordingly, a 12-month microbiological survey of water in the five ponds of the wetland commenced in August 2007. The aims of the survey are to determine changes, as effluent passes through the wetland system, in a broad range of indicator organisms (faecal coliforms, Escherichia coli, Enterococcus faecalis and Clostridium perfringens) and the occurrence of several pathogens - Salmonella, Campylobacter, Cryptosporidium and Mycobacterium avium subsp. paratuberculosis (Map). The highest indicator organism counts - E. coli and faecal coliforms, 103-104 CFU/ml - are observed in pond 1, and a significant reduction (1-3 log10) in all indicator organisms occurs as water passes through the wetland from pond 1 to pond 5. Hence the wetland is efficient at reducing levels of indicator organisms in the dairy effluent. Salmonella and Campylobacter spp. are being detected intermittently in all the ponds, whilst Cryptosporidium and Map have yet to be detected, and so the ability of the wetland to reduce/eliminate specific pathogens is less clear at present.

Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C35 extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild-type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid-producing bacteria.

Engineering plasmonic nanorod arrays for colon cancer marker detection

Engineering plasmonic nanomaterials or nanostructures towards ultrasensitive biosensing for disease markers or pathogens is of high importance. Here we demonstrate a systematic approach to tailor effective plasmonic nanorod arrays by combining both comprehensive numerical discrete dipole approximations (DDA) simulation and transmission spectroscopy experiments. The results indicate that 200×50 nm nanorod arrays with 300×500 nm period provide the highest FOM of 2.4 and a sensitivity of 310 nm/RIU. Furthermore, we demonstrate the use of nanorod arrays for the detection of single nucleotide polymorphism in codon 12 of the K-ras gene that are frequently occurring in early stages of colon cancer, with a sensitivity down to 10 nM in the presence of 100-fold higher concentration of the homozygous genotypes. Our work shows significant potential of nanorod arrays towards point-of-care applications in diagnosis and clinical studies.