Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry.

This review presents the results of a study into the offering of rapid microbial detection assays to the Irish dairy industry. At the outset, a consultation process was undertaken whereby key stakeholders were asked to compile a list of the key microorganisms of interest to the sector. The resultant list comprises 19 organisms/groups of organisms divided into five categories: single pathogenic species (Cronobacter sakazakii, Escherichia coli and Listeria monocytogenes); genera containing pathogenic species (Bacillus, Clostridium, Listeria, Salmonella; Staphylococcus); broad taxonomic groupings (Coliforms, Enterobacteriaceae, fecal Streptococci, sulfite reducing bacteria/sulfite reducing Clostridia [SRBs/SRCs], yeasts and molds); organisms displaying certain growth preferences or resistance as regards temperature (endospores, psychrotrophs, thermodurics, thermophiles); indicators of quality (total plate count, Pseudomonas spp.). A survey of the rapid assays commercially available for the 19 organisms/groups of organisms was conducted. A wide disparity between the number of rapid tests available was found. Four categories were used to summarize the availability of rapid assays per organism/group of organisms: high coverage (>15 assays available); medium coverage (5-15 assays available); low coverage (<5 assays available); no coverage (0 assays available). Generally, species or genera containing pathogens, whose presence is regulated-for, tend to have a good selection of commercially available rapid assays for their detection, whereas groups composed of heterogenous or even undefined genera of mainly spoilage organisms tend to be "low coverage" or "no coverage." Organisms/groups of organisms with "low coverage" by rapid assays include: Clostridium spp.; fecal Streptococci; and Pseudomonas spp. Those with "no coverage" by rapid assays include: endospores; psychrotrophs; SRB/SRCs; thermodurics; and thermophiles. An important question is: why have manufacturers of rapid microbiological assays failed to respond to the necessity for rapid methods for these organisms/groups of organisms? The review offers explanations, ranging from the technical difficulty involved in detecting as broad a group as the thermodurics, which covers the spores of multiple sporeforming genera as well at least six genera of mesophilic nonsporeformers, to the taxonomically controversial issue as to what constitutes a fecal Streptococcus or SRBs/SRCs. We review two problematic areas for assay developers: validation/certification and the nature of dairy food matrices. Development and implementation of rapid alternative test methods for the dairy industry is influenced by regulations relating to both the microbiological quality standards and the criteria alternative methods must meet to qualify as acceptable test methods. However, the gap between the certification of developer's test systems as valid alternative methods in only a handful of representative matrices, and the requirement of dairy industries to verify the performance of alternative test systems in an extensive and diverse range of dairy matrices needs to be bridged before alternative methods can be widely accepted and adopted in the dairy industry. This study concludes that many important dairy matrices have effectively been ignored by assay developers.

Mutagenic activities of biochars from pyrolysis.

Biochar production, from pyrolysis of lignocellulosic feedstocks, agricultural residues, and animal and poultry manures are emerging globally as novel industrial and commercial products. It is important to develop and to validate a series of suitable protocols for the ecological monitoring of the qualities and properties of biochars. The highly sensitive Salmonella mutagenicity assays (the Ames test) are used widely by the toxicology community and, via the rat liver extract (S9), can reflect the potential for mammalian metabolic activation. We examined the Ames test for analyses of the mutagenic activities of dimethylsulphoxide (DMSO) extracts of biochars using two bacterial models (S. typhimurium strains TA98 and TA100) in the presence and in the absence of the metabolic activation with the S9-mix. Tester strain TA98 was most sensitive in detecting mutagenic biochar products, and the contribution of S9 was established. Temperature and times of pyrolysis are important. Biochar pyrolysed at 400°C for 10min, from a lignocellulose precursor was mutagenic, but not when formed at 800°C for 60min, or at 600°C for 30min. Biochars from poultry litter, and manures of calves fed on grass had low mutagenicities. Biochar from pig manure had high mutagenicity; biochars from manures of cows fed on a grass plus cereals, those of calves fed on mother's milk, and biochars from solid industrial waste had intermediate mutagenicities. The methods outlined can indicate the need for further studies for screening and detection of the mutagenic residuals in a variety of biochar products.

Automatic decellularization of ovine aorta-derived extracellular matrix offers reduced processing and attendee times while being as effective as manual techniques.

OBJECTIVE: To construct an automatic decellularization platform (ADP) for preparing xenogenic extracellular matrices (ECMs), and to demonstrate that automatic decellularization for preparing xenogenic ECMs reduces processing time, requires fewer attendee hours, and is as effective as the manual gold standard preparation protocols. MATERIALS AND METHODS: A soft tissue ADP was constructed and ovine aorta was harvested (n=9). Manual and automatic decellularization was performed on aortic tissue specimens and both groups were compared. The presence of acellularity was assessed with viability/cytotoxicity assays, and the presence of residual ovine DNA was determined with gel electrophoresis and spectrophotometry. Scaffold integrity was characterized with scanning electron microscopy (SEM) and uniaxial tensile testing. RESULTS: Acellularity was confirmed with both preparation techniques and DNA concentrations measuring 540±130 and 590±270 ng/mg wet weight and the control measuring 6690±1210 ng/mg wet weight (p<0.05). SEM demonstrated no differences in the surface architecture of ECMs prepared by both techniques. Uniaxial testing demonstrated no significant differences in the incremental elastic moduli E below a stretch ratio of 2.70λ in both groups and a large reduction in E was recorded when both groups were compared with control samples above a stretch ratio of 1.7. CONCLUSION: Automatic decellularization of ovine aorta is as effective as gold standard manual decellularization protocols. Future research will focus on optimizing the automated decellularization technique and on upscaling protocols.

Cell-seeded extracellular matrices for bladder reconstruction: an ex vivo comparative study of their biomechanical properties.

PURPOSE: Autogenous ileal tissue remains the gold-standard biomaterial for bladder replacement purposes; however, cell-seeded extracellular matrix (ECM) scaffolds have shown promise. Although the biological advantages of cell-seeded ECMs in urological settings are well documented, there is a paucity of data available on their biomechanical properties. In this study, the biomechanical properties of cell-seeded ECMs are compared with autogenous ileal tissue. METHODS: Human urothelial cells (UCs) and smooth muscle cells (SMCs) were obtained by bladder biopsy and cultured onto porcine urinary bladder matrix (UBM) scaffolds under dynamic and static growth conditions for 14 days. The biomechanical properties of cell-seeded UBM (n = 12), and porcine ileum (n = 12) were determined with uni-axial tensile testing protocols and compared with stress-strain curves. In addition, their biomechanical properties were compared with porcine bladder tissue (n = 12) and unseeded UBM (n = 12). RESULTS: There were significant differences in the biomechanical properties of each biomaterial assessed. Strain to failure occurred at 92 ± 24% for dynamically cultured cell-seeded UBM compared to 42.2 ± 5.20% for ileal tissue (p<0.01). Values for linear stiffness at 30% strain were significantly lower in dynamically cultured cell-seeded UBM compared to ileal tissue (0.36 ± 0.14 MPa versus 0.67 ± 0.32 MPa respectively, p<0.01). Bladder tissue remained the most distensible biomaterial throughout, with linear stiffness measuring 0.066 ± 0.034 MPa at 30% strain. CONCLUSIONS: Dynamically cultured cell-seeded ECMs are biomechanically superior to ileal tissue for bladder replacement purposes. Additional comparative in vivo studies will be necessary before their role as a reliable alternative is clearly established.

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring.

PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency.

Phylogenetic analysis of the bacterial community in a full scale autothermal thermophilic aerobic digester (ATAD) treating mixed domestic wastewater sludge for land spread.

The bacterial community associated with a full scale autothermal thermophilic aerobic digester (ATAD) treating sludge, originating from domestic wastewater and destined for land spread, was analysed using a number of molecular approaches optimised specifically for this high temperature environment. 16S rDNA genes were amplified directly from sludge with universally conserved and Bacteria-specific rDNA gene primers and a clone library constructed that corresponded to the late thermophilic stage (t = 23 h) of the ATAD process. Sequence analyses revealed various 16S rDNA gene sequence types reflective of high bacterial community diversity. Members of the bacterial community included α- and ß-Proteobacteria, Actinobacteria with High G + C content and Gram-Positive bacteria with a prevalence of the Firmicutes (Low G + C) division (class Clostridia and Bacillus). Most of the ATAD clones showed affiliation with bacterial species previously isolated or detected in other elevated temperature environments, at alkaline pH, or in cellulose rich environments. Several phylotypes associated with Fe(III)- and Mn(IV)-reducing anaerobes were also detected. The presence of anaerobes was of interest in such large scale systems where sub-optimal aeration and mixing is often the norm while the presence of large amounts of capnophiles suggest the possibility of limited convection and entrapment of CO(2) within the sludge matrix during digestion. Comparative analysis with organism identified in other ATAD systems revealed significant differences based on optimised techniques. The abundance of thermophilic, alkalophilic and cellulose-degrading phylotypes suggests that these organisms are responsible for maintaining the elevated temperature at the later stages of the ATAD process.

Autothermal, single-stage, performic acid pretreatment of Miscanthus x giganteus for the rapid fractionation of its biomass components into a lignin/hemicellulose-rich liquor and a cellulase-digestible pulp.

A novel approach to the performic acid pulping of biomass enables effective delignification and fractionation in a time frame not achieved heretofore. An autothermal decomposition reaction was triggered when 100mg/L Fe(2)(SO(4))(3) in 4.0 M NaOH was added to 5% or 7.5% H(2)O(2) in aqueous formic acid containing chipped Miscanthus x giganteus. Peroxy-decomposition resulted in pressures of 19 and 35 bar in the 5% and 7.5% peroxide liquors and reduced the lignin content in the resulting pulps to <6% within 140 and 30 min, respectively. Solubilised lignin was available for recovery from the liquor by subsequent dilution with water. Hemicellulose removal to the liquor was 68% and 89% for the 5% and 7.5% peroxide solutions. Crystalline cellulose yields were >99% and >95% and the rate of glucose release from cellulase digestion of the pulps in 24h was more than 20-fold that for the raw Miscanthus.

Construction and evaluation of urinary bladder bioreactor for urologic tissue-engineering purposes.

OBJECTIVE: To design and construct a urinary bladder bioreactor for urologic tissue-engineering purposes and to compare the viability and proliferative activity of cell-seeded extracellular matrix scaffolds cultured in the bioreactor with conventional static growth conditions. MATERIALS AND METHODS: A urinary bladder bioreactor was designed and constructed to replicate physiologic bladder dynamics. The bioreactor mimicked the filling pressures of the human bladder by way of a cyclical low-delivery pressure regulator. In addition, cell growth was evaluated by culturing human urothelial cells (UCs) on porcine extracellular matrix scaffolds in the bioreactor and in static growth conditions for 5 consecutive days. The attachment, viability, and proliferative potential were assessed and compared with quantitative viability indicators and by fluorescent markers for intracellular esterase activity and plasma membrane integrity. Scaffold integrity was characterized with scanning electron microscopy and 4',6-diamidino-2-phenylindole staining. RESULTS: No significant difference in cell viability was identified between both experimental groups after 3 days of culture (P = .06). By day 4, the number of viable UCs was significantly greater in the bioreactor compared with the number cultured under static conditions (P = .009). A significant difference in UC viability was also present after 5 days of culture between the bioreactor and static group (P = .006). Viability/cytotoxicity assays performed on day 5 also confirmed the viability of UCs in both experimental groups. CONCLUSION: Significantly greater UC growth occurred on the extracellular matrix scaffolds cultured in the bioreactor compared with conventional static laboratory conditions after 3 days of culture. Our initial bioreactor prototype might be helpful for permitting additional advances in urinary bladder bioreactor technology.

Morphological characterisation of ATAD thermophilic sludge; sludge ecology and settleability.

Autothermal thermophilic aerobic digestion (ATAD) is a biological wastewater treatment process used for stabilisation of domestic, animal, food and pharmaceutical sludges, and wastewater. It produces a high-quality effluent due to thermophilic processing conditions, however the stabilised sludge has poor settling characteristics, a high water content, low compaction capacity and is difficult to dewater by mechanical processes alone. These factors impact transport and disposal of processed ATAD sludge. We have carried out a detailed morphological characterisation of ATAD sludge at all stages of the ATAD process in an attempt to determine key characteristics of the sludge that might be responsible for its poor dewatering and settleability. A number of microscopic techniques including electron, optical, wide field and laser scanning confocal microscopy were applied to fresh, fixed or embedded sludge taken at various stages during a full scale ATAD process treating domestic sludge. The spatial distributions of structural sludge matrix components were determined and suggested a highly dynamic sludge morphology during the overall process. Large amounts of fibres were observed in the feed sludge, whereas thermophilic sludge liquor with low settleability was shown to have a lower protein to polysaccharide ratio (1:0.9) compared to the easily settled fraction where ratio values were in the range of (1:1.14-1:1.7) with a prevalence of protein constituents. ATAD sludge was also shown to contain colloids, slime, cellulose micro-particles and multiple hydrophobic droplets in the bulk liquor, factors that may markedly impact on sludge dewaterability characteristics. Laser scanning confocal microscopy demonstrated a superior ability to identify composition and spatial localisation of structural constituents in such a dispersed, high water content sludge.

Evaluation of the removal of indicator bacteria from domestic sludge processed by Autothermal Thermophilic Aerobic Digestion (ATAD).

The degradation of sludge solids in an insulated reactor during Autothermal Thermophilic Aerobic Digestion (ATAD) processing results in auto-heating, thermal treatment and total solids reduction, however, the ability to eliminate pathogenic organisms has not been analysed under large scale process conditions. We evaluated the ATAD process over a period of one year in a two stage, full scale Irish ATAD plant established in Killarney and treating mixed primary and secondary sludge, by examining the sludge microbiologically at various stages during and following ATAD processing to determine its ability to eliminate indicator organisms. Salmonella spp. (pathogen) and fecal-coliform (indicator) densities were well below the limits used to validate class A biosolids in the final product. Enteric pathogens present at inlet were deactivated during the ATAD process and were not detected in the final product using both traditional microbial culture and molecular phylogenetic techniques. A high DNase activity was detected in the bulk sludge during the thermophilic digestion stage which may be responsible for the rapid turn over of DNA from lysed cells and the removal of mobile DNA. These results offer assurance for the safe use of ATAD sludge as a soil supplement following processing.

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry.

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.

C-NMR assessment of the pattern of organic matter transformation during domestic wastewater treatment by autothermal aerobic digestion (ATAD).

The pattern of biodegradation and the chemical changes occurring in the macromolecular fraction of domestic sludge during autothermal thermophilic aerobic digestion (ATAD) was monitored and characterised via solid-state (13)C-NMR CP-MAS. Major indexes such as aromaticity, hydrophobicity and alkyl/O-alkyl ratios calculated for the ATAD processed biosolids were compared by means of these values to corresponding indexes reported for sludges of different origin such as manures, soil organic matter and certain types of compost. Given that this is the first time that these techniques have been applied to ATAD sludge, the data indicates that long-chain aliphatics are easily utilized by the microbial populations as substrates for metabolic activities at all stages of aerobic digestion and serve as a key substrate for the temperature increase, which in turn results in sludge sterilization. The ATAD biosolids following treatment had a prevalence of O-alkyl domains, a low aromaticity index (10.4%) and an alkyl/O-alkyl ratio of 0.48 while the hydrophobicity index of the sludge decreased from 1.12 to 0.62 during the treatment. These results have important implications for the evolution of new ATAD modalities particularly in relation to dewatering and the future use of ATAD processed biosolids as a fertilizer, particularly with respect to hydrological impacts on the soil behaviour.

Vascular cell adhesion molecule-1 expression in endothelial cells exposed to physiological coronary wall shear stresses.

Atherosclerosis is consistently found in bifurcations and curved segments of the circulatory system, indicating disturbed hemodynamics may participate in disease development. In vivo and in vitro studies have shown that endothelial cells (ECs) alter their gene expression in response to their hemodynamic environment, in a manner that is highly dependent on the exact nature of the applied forces. This research exposes cultured ECs to flow patterns present in the coronary arterial network, in order to determine the role of hemodynamic forces in plaque initiation. Vascular cell adhesion molecule-1 (VCAM-1) was examined as an indicator of plaque growth, as it participates in monocyte adhesion, which is one of the initial steps in the formation of fatty lesions. The hemodynamics of a healthy right and left coronary artery were determined by reconstructing 3D models from cineangiograms and employing computational fluid dynamic models to establish physiological coronary flow patterns. Wall shear stress (WSS) profiles selected from these studies were applied to ECs in a cone and plate bioreactor. The cone and plate system was specifically designed to be capable of reproducing the high frequency harmonics present in physiological waveforms. The shear stresses chosen represent those from regions prone to disease development and healthier arterial segments. The levels of the transcriptional and cell surface anchored VCAM-1 were quantified by flow cytometry and real time RT-PCR over a number of timepoints to obtain a complete picture of the relationship between this adhesion molecule and the applied shear stress. The WSS profiles from regions consistently displaying a higher incidence of plaques in vivo, induced greater levels of VCAM-1, particularly at the earlier timepoints. Conversely, the WSS profile from a straight section of vessel with undisturbed flow indicated no upregulation in VCAM-1 and a significant downregulation after 24 h, when compared with static controls. Low shear stress from the outer wall of a bifurcation induced four times the levels of VCAM-1 messenger ribonucleic acid (mRNA) after four hours when compared with levels of mRNA induced by WSS from a straight arterial section. This shear profile also induced prolonged expression of the surface protein of this molecule. The current study has provided insight into the possible influences of coronary hemodynamics on plaque localization, with VCAM-1 only significantly induced by the WSS from disease prone regions.

ECM-based materials in cardiovascular applications: Inherent healing potential and augmentation of native regenerative processes.

The in vivo healing process of vascular grafts involves the interaction of many contributing factors. The ability of vascular grafts to provide an environment which allows successful accomplishment of this process is extremely difficult. Poor endothelisation, inflammation, infection, occlusion, thrombosis, hyperplasia and pseudoaneurysms are common issues with synthetic grafts in vivo. Advanced materials composed of decellularised extracellular matrices (ECM) have been shown to promote the healing process via modulation of the host immune response, resistance to bacterial infections, allowing re-innervation and reestablishing homeostasis in the healing region. The physiological balance within the newly developed vascular tissue is maintained via the recreation of correct biorheology and mechanotransduction factors including host immune response, infection control, homing and the attraction of progenitor cells and infiltration by host tissue. Here, we review the progress in this tissue engineering approach, the enhancement potential of ECM materials and future prospects to reach the clinical environment.

A novel ICE in the genome of Shewanella putrefaciens W3-18-1: comparison with the SXT/R391 ICE-like elements.

A novel R391-like ICE (integrating conjugative element) has been detected in the 4.2 MB genome of Shewanella putrefaciens W3-18-1 located on three different contigs. Assembly of the ICE encoding contigs based on similarity with R391 revealed a mosaic element of plasmid, phage and transposon-like sequences typical of SXT/R391 ICE-like elements. The element, which is 110 057 bp in length, was highly similar to R391 sequences, with most related ORFs showing >96% amino acid sequence identity. The element, designated ICESpuPO1, contained a number of inserts determining resistance to copper and other heavy metals and a broad-spectrum RND efflux pump similar to antibiotic efflux systems. The element was integrated into the Shewanella prfC gene in a manner similar to related ICE-like elements. The chromosomal element junctions contained a 17-bp SXT/R391-like attL and attR site and an unannotated ORF between attL and the ICE integrase encoding a putative recombinational directional factor necessary for excision, with 100% amino acid identity to the R391 ORF4 product.

Molecular tools to detect the IncJ elements: a family of integrating, antibiotic resistant mobile genetic elements.

The IncJ group of enterobacterial mobile genetic elements, which include R391, R392, R705, R997 and pMERPH, have been shown to be site-specific integrating elements encoding variable antibiotic and heavy metal resistance genes. They insert into a specific 17-bp site located in the prfC gene, encoding peptide release factor 3, in Escherichia coli and other hosts. A key feature of known IncJ elements is the presence of a site-specific recombination module consisting of an attachment site on the element and an integrase-encoding gene of the tyrosine recombinase class, which promotes integration between the attachment site on the element and a similar site on the host chromosome. We have cloned and sequenced the integrases from a number of known IncJ elements and designed PCR primers for specific amplification of this gene. Using conserved regions of enterobacterial prfC genes upstream and downstream of the insertion site, and conserved sequences at the ends of the integrated IncJ elements, we have designed specific primers to amplify across the integrated IncJ attL and attR junction fragments. Alignment of over 30 enterobacterial prfC-like genes indicates that the primers designed to amplify attR junction would amplify IncJ element: host junctions from a wide variety of hosts. The IncJ elements have been shown to sensitise recA(+)E. coli K12 strains to UV irradiation. A simple and rapid procedure for demonstrating this effect is described. These tools should enable the rapid detection of such elements in clinical and environmental settings.

Detection and typing of Borrelia burgdorferi sensu lato genospecies in Ixodes persulcatus ticks in West Siberia, Russia.

The prevalence of Borrelia burgdorferi sensu lato (s.l.) genospecies in West Siberia as well as in many other regions of Russia remains insufficiently investigated. In the present study a total of 151 adult female ticks Ixodes persulcatus Schulze, collected at three localities in eastern regions of West Siberia, where Lyme disease is endemic, were examined for the presence of the spirochete B. burgdorferi s.l. by polymerase chain reaction targeting the 23S-5S rRNA intergenic spacer regions. Spirochetal DNA was detected in on average 15.2+/-3.0% of the ticks examined. The infection rate of adult ticks with B. burgdorferi s.l. at various localities ranged from 8.6+/-3.4% to 29.0+/-7.6%, being greatest in the northernmost site studied and decreasing southwards. The restriction patterns obtained after MseI digestion of the 23S-5S rRNA intergenic spacer amplicons assigned 23 DNA samples to the following genomic groups: 19 to B. garinii (12 to group NT29 and seven to group 20047(T)), three to B. afzelii, and one to mixed B. afzelii and B. garinii NT29. We have not detected other genospecies, which were found in ticks in Europe, the Russian Far East and Japan. Thus, the ticks examined were associated only with two genospecies of Borrelia burgdorferi s.l. pathogenic to humans (B. garinii and B. afzelii), and B. garinii was the major genospecies infecting adult I. persulcatus in eastern regions of West Siberia.