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A PCR-based assay helps identify Bacillus cereus in foods. Experimental studies help design three oligonucleotide primers within the sequence for cereolysin AB, a cytolytic determinant encoding hemolytic and lecithin-hydrolyzing activities of B. cereus. Examination of the specificity of these primers using a combined PCR-hybridization assay reveals the significant specificity of these primers for isolates of the B. cereus group. This combined assay helps detect B. cereus in artificially contaminated milk and the detection limit is 1 CFU ml of milk.

Consistent individual differences in behavior [i.e., behavioral types (BTs)], are common across the animal kingdom. Consistency can make behavior an adaptive trait for mate choice decisions. Here, we present a conceptual framework to explain how and why females might evaluate a male's BT before mating. Because BTs are consistent across time or context, a male's BT can be a reliable indicator of his potential to provide direct benefits. Heritable BTs can enable informed mate choice via indirect benefits. Many key issues regarding patterns of mate choice, including sensory biases, context dependence, and assortative mating apply to BT-dependent mate choice. Understanding the relationship between BTs and mate choice may offer insights into patterns of variation and consistency common in behavioral traits., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The increasing lack of drinking water around the globe is of great concern. Although UV irradiation, photocatalysis, and electrocatalysis for bacterial disinfection have been widely explored, the synergistic kinetics involved in these strategies have not been reported to date. Herein, we report on an efficient and cost-effective strategy for the remediation of a model bacterium (E. coli), through the integration of photochemistry and electrochemistry based on a bifunctional electrode, which utilizes titanium (Ti) as the substrate, nanoporous titanium dioxide (TiO 2 ) as a photocatalyst, and ruthenium oxide (RuO 2 ) nanoparticles as an electrocatalyst. The nanoporous TiO 2 was grown directly onto a Ti substrate via a three-step anodization process, and its photocatalytic activity was significantly enhanced by a facile electrochemical treatment. A high disinfection rate at 0.62 min -1 , with >99.999% bacterial removal within 20 min was achieved using the novel TiO 2 /Ti/RuO 2 bifunctional electrode. Complete bacterial disinfection was attained within 30 min as assessed by a spread plate method. Bacterial survival strategies, including a viable but non-culturable state of the bacteria, were also investigated during the bifunctional treatment process. The novel strategy demonstrated in this study has strong potential to be utilized for water purification and wastewater treatment as an advanced environmentally compatible technology., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Entrapment is one of the major approaches for enzyme immobilization; however, it suffers a few critical drawbacks including leakage and high mass transfer resistance to substrates. To address these challenges, herein we report on a new facile and effective enzyme entrapment platform using a special cationic polymer, poly(2-(dimethylamino)ethyl methacrylate) (MADQUAT) on a single-wall carbon nanotube and reduced graphene oxide (SWCNT-rGO) nanohybrid thin film. To demonstrate this new approach, alcohol dehydrogenase (ADH) is employed as a model enzyme for the entrapment toward the design of an efficient electrochemical biosensor for the detection of ethanol. MADQUAT possesses strong electrostatic affinity with various negatively charged biomolecules; and our FTIR study has shown that there are no structural changes in the enzyme following the entrapment, with an excellent secondary structure association (r = 0.92). Our electrochemical measurements have shown that the entrapped ADH exhibits high ability to exchange electrons in the presence of the NAD + /NADH cofactor and that the SWCNT-rGO nanohybrid significantly enhances the biocatalytic activity of the immobilized ADH and the electrochemical oxidation of NADH in comparison with either SWCNTs or rGO. The ethanol biosensor developed in this study exhibits a fast response, wide linearity range, high sensitivity (26.27 μA mM -1 cm -2 ), remarkable low limit of detection (0.16 μM), high selectivity and high stability. The optimized biosensor has been further tested with real samples including wine, beer and blood alcohol, showing promising analytical and biomedical applications.

Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with Bac Light biovolume analysis and conventional plate counting for the enumeration of C. jejuni-removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or Bac Light biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the Bac Light assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 microg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt+ve and -ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H(2)O(2)-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed.

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and beta-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.

In this study, we use thermodynamic theory to develop a novel model that allows for the quantitative determination of the Gibbs free energy of adhesion for the initial bacterial attachment process. This model eliminates the need to calculate interfacial free energies and instead relies on easily measurable contact angles to determine DeltaG(adh). We experimentally verify our model using real-time observation of the initial attachment of Pseudomonas putida to methyl- and hydroxyl-terminated self-assembled monolayers. We also test the applicability of our model to a variety of experimental conditions using data available in the literature. We show that the initial attachment process is governed by dispersion forces and is accurately predicted by our model. Also, we find that our model is simple to apply and accurate for a variety of experimental conditions.

The role of stationary phase sigma factor gene (rpoS) in the stress response of Moraxella strain when exposed to radiation was determined by comparing the stress responses of the wild-type (WT) and its rpoS knockout (KO) mutant. The rpoS was turned on by starving the WT cultures for 24 h in minimal salt medium. Under non-starved condition, both WT and KO planktonic Moraxella cells showed an increase in mortality with the increase in duration of irradiation. In the planktonic non-starved Moraxella, for the power intensity tested, UV radiation caused a substantially higher mortality rate than did by the visible laser light (the mortality rate observed for 15-min laser radiation was 53.4 +/- 10.5 and 48.7 +/- 8.9 for WT and KO, respectively, and 97.6 +/- 0 and 98.5 +/- 0 for 25 s of UV irradiation in WT and KO, respectively). However, the mortality rate decreased significantly in the starved WT when exposed to these two radiations. In comparison, rpoS protected the WT against the visible laser light more effectively than it did for the UV radiation. The WT and KO strains of Moraxella formed distinctly different types of biofilms on stainless steel coupons. The KO strain formed a denser biofilm than did the WT. Visible laser light removed biofilms from the surfaces more effectively than did the UV. This was true when comparing the mortality of bacteria in the biofilms as well. The inability of UV radiation to penetrate biofilms due to greater rates of surface absorption is considered to be the major reason for the weaker removal of biofilms in comparison to that of the visible laser light. This result suggests that high power visible laser light might be an effective tool for the removal of biofilms., ((c) 2006 Wiley Periodicals, Inc.)

Seven strains of Listeria monocytogenes frequently involved in foodborne disease (epidemic strains) and 14 sporadic strains were examined to compare the attachment and subsequent biofilm growth on glass slides at 37 degrees C. Epidemic strains at 3 h incubation had significantly higher attachment values than sporadic strains (P<0.001), but subsequent biofilm growth over 24 h was not dependent on initial attachment. To better understand this phenomenon, the surface hydrophobicity and charge, as well as the extracellular carbohydrate content of the 21 L. monocytogenes strains were studied to determine if these surface characteristics had an effect on bacterial attachment to glass. Hydrophobicity was measured by the bacterial adherence to hydrocarbon (BATH) and polystyrene adherence methods. Hydrophobicity values obtained with the BATH method were linearly correlated with those from the polystyrene adherence method (r=0.64, P<0.001), but no correlation was found between hydrophobicity and bacterial attachment to glass. Hydrophobicity and surface charge measured as electrophoretic mobility (EM) were correlated (r=0.77, P<0.001); however, there was no correlation between the degree of attachment and surface charge. Colorimetric measurements of the total extracellular carbohydrates revealed that attached cells produced significantly (P<0.05) higher levels than planktonic cells after a 3 h time period. Analysis of co-variance (Nested ANCOVA) furthermore demonstrated that total carbohydrates produced by planktonic cells had a significant positive effect on 24 h biofilm growth (P=0.006). This is the first report to indicate that the ability of a L. monocytogenes strain to produce high levels of extracellular carbohydrates may increase its ability to form a biofilm. Genetic studies targeting carbohydrate synthesis pathways of L. monocytogenes will be required to fully understand the importance of this observation.

A multiplex PCR-DNA probing assay was developed to detect four major Escherichia coli virotypes. Six highly specific polymerase chain reaction (PCR) primer sets and DIG-labeled chemiluminescent probes were designed to target the Shiga-like toxin I and II genes (stxI and stxII) of verotoxigenic E. coli (VTEC), heat-stable and heat-labile toxin genes of enterotoxigenic E. coli (ETEC), adherence factor (EAF) of enteropathogenic E. coli (EPEC) and a fragment of the invasiveness plasmid (IAL) of enteroinvasive E. coli (EIEC). The primer pairs generate products of 350, 262, 170, 322, 293 and 390 bp in length, respectively. The multiplex primers and probes were tested for specificity against 31 pathogenic E. coli strains, nine nonpathogenic E. coli and non-E.coli enteric and environmental bacterial strains. The results showed a high degree of specificity of the primers and probes for strains from corresponding virotypes and no reaction with the nontarget bacterial strains. The proposed multiplex PCR-DNA probing assay provides rapid and specific detection of four major virotypes of E. coli.

The extraction of DNA from manure and the subsequent polymerase chain reaction (PCR) amplification of virulence genes to detect pathogens require an effective method of purification. Four different methods were assessed for their effectiveness in extracting and purifying Escherichia coli O157:H7 DNA from cattle manure: phenol/chloroform purification, phenol/chloroform/Sepharose B4 spin columns, phenol/chloroform/polyvinylpolypyrrolidone (PVPP) spun columns, and Mo Bio UltraClean kit. A PCR assay targeting the shiga-like toxin I gene (sltI) was carried out to determine the effectiveness of the four methods in removing PCR inhibitors from the manure samples. All methods were used to extract a manure slurry and the cleanliness of the samples was tested by the PCR with varying concentrations of spiked E. coli O157:H7 target DNA. The PVPP spun columns and the UltraClean kit had the best detection limit, detecting 20 pg of E. coli DNA (about 2x10(3) cells) per 100 mg of manure. The UltraClean kit and the PVPP spun columns also had the best and similar detection limits of 3x10(4) CFU/100 mg manure when E. coli O157:H7 cells were spiked into the manure sample and purified by all four methods. The enrichment of cells after inoculation into manure was performed using tryptic soy broth at 37 degrees C for 5 h. Both the PVPP spun columns and the UltraClean kit methods were used to purify the enriched samples and were able to detect initial inocula of 6 CFU/100 mg manure, indicating that the two methods were highly efficient in purifying DNA from manure samples.

Production liability, safety regulations as well as marketing strategies are important motivations for companies to establish in-house quality control measures which can be certified by an independent control body. Companies that advertise quality control measures place pressure on opposition companies to follow suite. It follows that certification in foodstuff production will lead to a chain reaction and spread quickly to other companies. In the establishment of a quality assurance system it is just as important to distinguish between quality control and quality assurance as it is to consider the guidelines laid down in the ISO-Standards 9000-9004. After certification according to the ISO-Standards, which dictates certain forms of quality assurance, there is no guarantee that all important risks, such as hygiene risks introduced with technology, can be excluded. In terms of public health monitoring an important responsibility awaits veterinary public health. Strategies and consequences for official monitoring of foodstuffs are presented and discussed.

Swab specimens from 4357 pig hindquarters provided for production of cured raw ham were contaminated with Staphylococcus aureus in 22.7%. The bacterial counts for S. aureus on the rind surface of the raw, uncured ham were between 10(1) and 10(3) cfu/cm2 in 89% of the positive samples. In the remaining 11% of contaminated ham counts of 10(3) to 10(6) cfu/cm2 were determined. There were major differences in the rate of contamination between pork from different suppliers. Questioning the suppliers revealed that pork highly contaminated with S. aureus could be traced back to certain abattoirs. This suggests that the technique of slaughter, concomitant hygiene precautions and the subsequent refrigeration of the carcasses affect the contamination of the meat with staphylococci.