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The efficiency with which E.coli BL21 can be modified using CRISPR-Cas9 genetic engineering is several orders of magnitude lower than that of E. coli W3110. We show that the lack of Lon protease is responsible, and demonstrate that restoration of the Lon protease or knock-out of sulA improves CRISPR-Cas9 engineering efficiency of BL21 to levels comparable to E. coli W3110.

As the first commercially available surface plasmon resonance (SPR) device, the BIAcore was primarily conceived with the intention of analyzing macromolecular interactions involving reasonably well defined, purified and soluble interactants. While this may be experimentally optimal it is not always possible nor desirable in a biological context. Many critical biological interactions, for example signaling events, occur at the cell surface between a membrane-bound receptor and a soluble ligand, or between two membrane-bound receptors on different cells. In many instances lipids are a requirement for the biological activity of a receptor (Haring et al., 1991) or they may even be the interactant itself (Kuziemko et al., 1996). Furthermore, membrane-associated receptors may not be soluble in an aqueous medium in the absence of detergent (Elliot and Raftery, 1979) and as a consequence, are not amenable to chemical immobilization or result in a dysfunctional immobilized ligand. In these situations, working with lipid structures may be a matter of necessity. On a more basic level, having to work with membranes may be simply due to the non-availability of purified receptors or that such receptors have yet to be identified.

Ligand-activated signaling through the type 1 insulin-like growth factor receptor (IGF1R) is implicated in many physiological processes ranging from normal human growth to cancer proliferation and metastasis. IGF1R has also emerged as a target for receptor-mediated transcytosis, a transport phenomenon that can be exploited to shuttle biotherapeutics across the blood–brain barrier (BBB). We employed differential hydrogen–deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) to characterize the interactions of the IGF1R ectodomain with a recently discovered BBB-crossing single-domain antibody (sdAb), VHH-IR5, in comparison with IGF-1 binding. HDX-MS confirmed that IGF-1 induced global conformational shifts in the L1/FnIII-1/-2 domains and α-CT helix of IGF1R. In contrast, the VHH-IR5 sdAb-mediated changes in conformational dynamics were limited to the α-CT helix and its immediate vicinity (L1 domain). High-resolution NMR spectroscopy titration data and linear peptide scanning demonstrated that VHH-IR5 has high-affinity binding interactions with a peptide sequence around the C-terminal region of the α-CT helix. Taken together, these results define a core linear epitope for VHH-IR5 within the α-CT helix, overlapping the IGF-1 binding site, and suggest a potential role for the α-CT helix in sdAb-mediated transcytosis.

The rapid detection of foodborne microbial pathogens contaminating fresh fruits and vegetables during the intervening period between harvest and consumption could revolutionize microbial quality assurance of food usually consumed raw and those with a limited shelf life. We have developed a sensitive, shotgun whole genome sequencing protocol capable of detecting as few as 1 colony forming unit (cfu) of Salmonella enterica serovar Typhimurium spiked on 25 g of lettuce. The Ion Torrent sequencing platform was used to generate reads of globally amplified DNA from microbes recovered from the surface of lettuce followed by bioinformatic analyses of the nucleotide sequences to detect the presence of Salmonella. The test is rapid and sensitive, and appropriate for testing perishable foods, and those consumed raw, for Salmonella contamination. The test has the potential to be universally applicable to any microbial contaminant on lettuce as long as a suitable bioinformatics pipeline is available and validated. A universal test is expected to pave the way for preventive and precision food safety and the re-shaping of the entire spectrum of food safety investigations from the current disease-limiting, reactive procedure to a proactive, disease prevention process.

The rapid detection of foodborne microbial pathogens contaminating fresh fruits and vegetables during the intervening period between harvest and consumption could revolutionize microbial quality assurance of food usually consumed raw and those with a limited shelf life. We have developed a sensitive, shotgun whole genome sequencing protocol capable of detecting as few as 1 colony forming unit (cfu) of Salmonella enterica serovar Typhimurium spiked on 25 g of lettuce. The Ion Torrent sequencing platform was used to generate reads of globally amplified DNA from microbes recovered from the surface of lettuce followed by bioinformatic analyses of the nucleotide sequences to detect the presence of Salmonella. The test is rapid and sensitive, and appropriate for testing perishable foods, and those consumed raw, for Salmonella contamination. The test has the potential to be universally applicable to any microbial contaminant on lettuce as long as a suitable bioinformatics pipeline is available and validated. A universal test is expected to pave the way for preventive and precision food safety and the re-shaping of the entire spectrum of food safety investigations from the current disease-limiting, reactive procedure to a proactive, disease prevention process.

Many plants have natural partnerships with microbes that can boost their nitrogen (N) and/or phosphorus (P) acquisition. To assess whether wheat may have undiscovered associations of these types, we tested if N/P-starved Triticum aestivum show microbiome profiles that are simultaneously different from those of N/P-amended plants and those of their own bulk soils. The bacterial and fungal communities of root, rhizosphere, and bulk soil samples from the Historical Dryland Plots (Lethbridge, Canada), which hold T. aestivum that is grown both under N/P fertilization and in conditions of extreme N/P-starvation, were taxonomically described and compared (bacterial 16S rRNA genes and fungal Internal Transcribed Spacers - ITS). As the list may include novel N- and/or P-providing wheat partners, we then identified all the operational taxonomic units (OTUs) that were proportionally enriched in one or more of the nutrient starvation- and plant-specific communities. These analyses revealed: a) distinct N-starvation root and rhizosphere bacterial communities that were proportionally enriched, among others, in OTUs belonging to families Enterobacteriaceae, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Cytophagaceae, Streptomycetaceae, b) distinct N-starvation root fungal communities that were proportionally enriched in OTUs belonging to taxa Lulworthia, Sordariomycetes, Apodus, Conocybe, Ascomycota, Crocicreas, c) a distinct P-starvation rhizosphere bacterial community that was proportionally enriched in an OTU belonging to genus Agrobacterium, and d) a distinct P-starvation root fungal community that was proportionally enriched in OTUs belonging to genera Parastagonospora and Phaeosphaeriopsis. Our study might have exposed wheat-microbe connections that can form the basis of novel complementary yield-boosting tools.

This study describes the application of the multivariate curve resolution (MCR) analysis technique for real-time analysis of culture fluorescence during recombinant Pichia pastoris cultivation in a bioreactor. Fluorescence spectra were acquired with an on-line dual excitation wavelength fluorometer and then used to develop a real time MCR-based bioprocess monitoring and diagnostics tool. Initial bioreactor experiments using two similar recombinant antibody secreting P. pastoris cell lines showed significant differences in protein production. To distinguish between the contributions of operating conditions and the specific cell line's genetic composition to the observed differences in protein production, the bioreactor experiments were repeated and accompanied by real time MCR analysis. The tests demonstrated high sensitivity of MCR-derived "pure concentration" profiles to growth as well as to initial conditions, thus enabling real-time cultivation process trend diagnostics and fault detection. © 2018 Her Majesty the Queen in Right of Canada © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2761, 2019.

We investigated whether specific sequence types, and their shared virulence gene profiles, may be associated with both human and food animal reservoirs. A total of 600 Escherichia coli isolates were assembled from human (n=265) and food-animal (n=335) sources from overlapping geographic areas and time periods (2005-2010) in Canada. The entire collection was subjected to multilocus sequence typing and a subset of 286 E. coli isolates was subjected to an E. coli-specific virulence gene microarray. The most common sequence type (ST) was E. coli ST10, which was present in all human and food-animal sources, followed by ST69, ST73, ST95, ST117, and ST131. A core group of virulence genes was associated with all 10 common STs including artJ, ycfZ, csgA, csgE, fimA, fimH, gad, hlyE, ibeB, mviM, mviN, and ompA. STs 73, 92, and 95 exhibited the largest number of virulence genes, and all were exclusively identified from human infections. ST117 was found in both human and food-animal sources and shared virulence genes common in extraintestinal pathogenic E. coli lineages. Select groups of E. coli may be found in both human and food-animal reservoirs.

After the introduction of the glycoconjugate vaccine based upon the capsular polysaccharide of Haemophilus influenzae type b in the mid 1980s there was a remarkable decrease in the number of invasive cases reported for this organism. Since the 1990s several groups have observed the emergence of Haemophilus influenzae type a (Hia), especially in indigenous communities in the northern regions of Canada and Alaska, to a stage where a solution is warranted to prevent further unnecessary deaths due to this pathogen. A glycoconjugate vaccine solution based upon the type a capsular polysaccharide (CPS) was investigated pre-clinically in an effort to illustrate the proof of concept for this approach. In this study we describe the growth of Hia and the isolation, purification and conjugation of the CPS to several carrier proteins. The resulting glycoconjugates were immunised in mice and rabbits provoking sera that facilitated bactericidal killing against all type a strains that we tested. This study has illustrated the pre-clinical proof of concept of a glycoconjugate vaccine based on the CPS of Hia as a solution to this emerging disease.

International audience; A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.

The objective of this study was to assess the antimicrobial resistance and virulence genotypes of Enterococcus faecalis isolated from samples obtained from a commercial pork processing plant. A total of 200 samples were randomly obtained from carcasses after bleeding (BC; 50 samples) and pasteurization (PC; 100 samples) and from retail pork products (RP; 50 samples). One isolate from each E. faecalis -positive sample was analyzed for antimicrobial susceptibility and characterized using a enterococcal microarray for analysis of resistance and virulence genes. E. faecalis was isolated from 79.5% of BC samples, 2% of PC samples, and 72.7% of RP samples. Resistance to the clinically important drugs ciprofloxacin (one isolate each from BC and RP samples) and daptomycin (one isolate each from PC and RP samples) was found. Multiresistance (to five or more antimicrobials) was more common in E. faecalis isolates from BC (77.4% of isolates) samples than those from PC (25%) and RP (37.6%) samples. Resistance to kanamycin (43.5%) and streptomycin (69.2%) was noted mostly in E. faecalis from BC samples. The most common resistance genes (5% prevalence) found in E. faecalis were those for aminoglycosides (aac(6), aphA3, and aadE), macrolides-lincosamide (ermB, ermA, sat(4), and linB), and tetracyclines (tetL, tetM, and tetO ). The virulence genes expressing adhesion (ace, efaAfs, and agrBfs), gelatinase (gelE), and pheromone (cAM, ccF10, cob, and cpd1) factors were found in the majority of isolates. Significant associations were found between resistance and virulence genes, suggesting their possible relationship. These data suggest that carcasses entering the final product processing area are mostly free of E. faecalis but are recontaminated with antimicrobial-resistant strains during processing. The source of these contaminants remains to be identified; however, these results underscore the importance of E. faecalis as a reservoir of resistance and virulence genes.

From the Camelidae family members, several serotypes of Escherichia coli (E. coli) have recently been isolated from diarrhoeic and non-diarrhoeic faecal samples. To date Shiga toxin-producing E. coli (STEC) strains have never been typed in one-humped camel (Camelus dromedarius). In the present study, two E. coli O157:H7 strains isolated from sick dromedaries were investigated. Virulence gene profiles were determined using a custom E. coli virulence DNA microarray, composed of 70-mer oligonucleotide probes targeting 264 virulence or related genes of known E. coli pathotypes. Both strains displayed positive hybridization signals for the Locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized in STEC.

A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA , recA , pheS , and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium , E. faecalis , E. durans , E. gallinarum , and E. avium . The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae . Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential.

As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Quebec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.

The use of DNA-based molecular detection tools for bacterial diagnostics is hampered by the inability to distinguish signals originating from live and dead cells. The detection of live cells is typically most relevant in molecular diagnostics. DNA-intercalating dyes like ethidium monoazide and propidium monoazide (PMA) offer a possibility to selectively remove cells with compromised cell membranes from the analysis. Once these dyes enter a cell, they bind to DNA and can be covalently crosslinked to it by light exposure. PCR amplification of such modified DNA is strongly inhibited. In this study we evaluated the suitability of propidium monoazide treatment to exclude isopropanol-killed cells from detection in defined mixtures using diagnostic microarray technology. The organisms comprised Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Serratia marcescens, and Escherichia coli O157:H7. PCR products obtained from amplification of chaperonin 60 genes (cpn60; coding for GroEL) were hybridized to a custom-designed microarray containing strain-specific cpn60-based 35-mer oligonucleotide probes. Results were compared with data from quantitative PCR, which confirmed that PMA could successfully inhibit amplification of DNA from killed cells in the mixtures. Although microarray data based on analysis of end-point PCR amplicons is not quantitative, results showed a significant signal reduction when targeting killed cells and consistently agreed with qPCR results. Treatment of samples with PMA in combination with diagnostic microarray detection can therefore be considered beneficial when analyzing mixtures of intact and membrane-compromised cells. Minimization of detection signals deriving from dead cells will render data more relevant in studies including pathogen risk assessment.

We used suppression subtractive hybridization (SSH) and differential screening (DS) to generate cDNA libraries enriched for up-regulated host and pathogen genes following inoculation of velvetleaf leaves with the mycoherbicide Colletotrichum coccodes. A total of 116 expressed sequence tags (EST), representing preferentially expressed genes, were analyzed, of which 89 sequences were assigned putative functions including transcription and translation, energy, cell growth and maintenance, and oxidative stress and defense. The EST sequences were assembled into 20 contigs and 29 singletons/singlets. Except for contig 26 that had the highest hit to fungal Histone 4, all other sequences had top hits to plant sequences. Genes related to oxidative stress and defense formed the largest category corresponding to nine contigs and three singlets (or 48% of the ESTs). The metallothioneins (MT) type 3 proteins were represented by six contigs, three singletons, and two singlets. We also identified genes for the ethylene response element binding (EREB) protein, WRKY, and bZIP proteins, which are likely to play roles in transcription, as well as genes for proteins such as ascorbate peroxidase and reticuline oxidase. The up-regulation of seven selected genes were validated by quantitative real-time PCR. Relative to uninfected velvetleaf leaves, these genes were significantly induced by the presence of C. coccodes on velvetleaf leaves.

Mycoparasitism by antagonistic fungi involves changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during mycoparasite– host interaction represents a powerful strategy to obtain insight into the molecular events underlying these changes. The aim of this study is to identify genes whose expression is upregulated when the mycoparasite Stachybotrys elegans is in direct confrontation with its host Rhizoctonia solani. Suppression subtractive hybridization (SSH) was used to create a subtracted cDNA library, and differential screening was applied to identify the over-expressed transcripts. We report the analysis of 2,166 clones, among which 47% were upregulated during mycoparasitism. Two hundred and sixtyone clones were sequenced that corresponded to 94 unique genes. Forty-four of these were identified as novel genes, while the remainder showed similarity to a broad diversity of genes with putative functions related to toxin production, pathogenicity, and metabolism. As a result of mycoparasitism, 15 genes belonged to R. solani among which 9 genes were assigned putative functions. Quantitative RT-PCR was used to examine the upregulation of 12 genes during the course of mycoparasitism. Seven genes showed significant upregulation at least at one-time point during interaction of the mycoparasite with its host. This study describes a first step toward knowledge of S. elegans genome. The results present the useful application of EST analysis on S. elegans and provide preliminary indication of gene expression putatively involved in mycoparasitism.

The virulence gene profile of 26 rabbit enteropathogenic Escherichia coli strains, isolated from 17 colibacillosis outbreaks located in two regions of Northern Italy, was determined using an Echerichia coli virulence DNA microarray. All strains were classified according to their determined biotype, sero- and phylo-group. The distribution of virulence genes encoding for the Locus of enterocyte effacement (LEE), LEE type III secretion system (T3SS), non-LEE T3SS translocated proteins and adherence factors was also determined. All strains but one belonged to phylogroups A and B1. A prevalent association between the O103 serogroup with the rhamnose-negative phenotype (biotype 12 or 14) was found. The most prevalent LEE profile found in tested strains was ler/cesT/espA-1/espB-3/tir-1/eae(beta)/espD-2/escN/eprJ. All strains possessed either the adhesive factor rabbit-2 (afr/2) or the plasmid Rabbit adherence locus (ral) gene and 24 of them an additional individual or combined set of colonization factors efa1/lifA, lpfA and paa genes. Finally, the combined or single presence of a set of LEE and/or non-LEE effector proteins encoding genes, namely espG, cif, map and nle family genes, attested to the genetic potential of investigated strains to induce pathologic lesions to the host. The application of microarray-based technologies in assessing the genetic profile of rabbit E. coli is a reliable, cost-effective candidate for large scale investigations in monitoring programs aimed to survey the circulation of pathogenic strains within rabbit production units, their zoonotic genetic potential and to select E. coli strains eligible for vaccinal prophylaxis in fattening rabbit production.

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant “pyramids” producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses.

Fecal contamination of surface waters is one the major sources of waterborne pathogens and consequently, is an important concern for public health. For reliable fecal source tracking (FST) monitoring, there is a need for a multi-marker toolbox as no single all-encompassing method currently exists. Mitochondrial DNA (mtDNA) as a source tracking marker has emerged as a promising animal-specific marker. However, very few comprehensive field studies were done on the occurrence of this marker in surface waters. In this report, water samples were obtained from 82 sites in different watersheds over a six year period. The samples were analyzed for the presence of human, bovine and porcine mtDNA by endpoint nested PCR, along with the human-specific Bacteroidales HF183 marker. These sites represented a mix of areas with different anthropogenic activities, natural, urban and agricultural. The occurrences of mitoHu (human), mitoBo (bovine), mitoPo (porcine) and HF183 specific PCR amplifications from the samples were 46%, 23%, 6% and 50%, respectively. The occurrence of mitoHu and HF183 was high in all environment types with higher occurrence in the natural and urban areas, whereas the occurrence of mitoBo was higher in agricultural areas. FST marker concentrations were measured by real-time PCR for samples positive for these markers. The concentration of the mitoHu markers was one order of magnitude lower than HF183. There was co-linearity between the concentrations of the mitoHu and HF183 markers. Co-linearity was also observed between HF183 concentration and fecal coliform levels. Such a relationship was not observed between the mitoHu concentration and fecal coliform levels. In summary, our results showed a high incidence of human fecal pollution throughout the environment while demonstrating the potential of mtDNA as suitable FST markers.

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a 'splash'-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin–horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.

Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.Keywords: Bacterial DNA extraction; Pathogen detection; Microarrays; Wastewater

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100% mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21% of the total soluble proteins. Bioassays with R1 transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.

DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.

Public concern over polluted water is a major environmental issue worldwide. Microbial contamination of water arguably represents the most significant risk to human health on a global scale. An important challenge in modern water microbial quality monitoring is the rapid, specific, and sensitive detection of microbial indicators and waterborne pathogens. Presently, microbial tests are based essentially on time-consuming culture methods. Rapid microbiological analyses and detection of rare events in water systems are important challenges in water safety assessment since culture methods present serious limitations from both quantitative and qualitative points of view. To circumvent lengthy culture methods, newer enzymatic, immunological, and genetic methods are being developed as an alternative. DNA microarray technology is a new and promising tool that allows the detection of several hundred or even thousands DNA sequences simultaneously. Recent advances in sample processing and DNA microarray technologies provide new perspectives to assess microbial water quality. The aims of this review are to (1) summarize what is currently known about microbial indicators, (2) describe the most important waterborne pathogens, (3) present molecular methods used to monitor the presence of pathogens in water, and (4) show the potential of DNA microarrays in water quality monitoring.

A total of 112 Escherichia coli O149:K91 strains isolated from pigs with diarrhea in Quebec, Canada, between 1978 and 2000 were characterized for their genotypic antimicrobial resistance profiles. Tests for resistance to 10 antimicrobial agents were conducted. Resistance to tetracycline and sulfonamides was found to be the most frequent, but resistance to cefotaxime and ceftiofur was absent. An increase in the number of isolates resistant to at least three antimicrobials was observed over time. The distribution of 28 resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, tetracycline, trimethoprim, and sulfonamides) was assessed by colony hybridization. Significant differences in the distributions of tetracycline [ tet (A), tet (B), tet (C)], trimethoprim ( dhfrI , dhfrV , dhfrXIII ), and sulfonamide ( sulI , sulII ) resistance genes were observed during the study period (1978 to 2000). Sixty percent of the isolates possessed a class 1 integron, illustrating the importance of integrons in the epidemiology of antibiotic resistance in E. coli strains from pigs. Amplification of the integron's variable region resulted in four distinct fragments of 1, 1.3, 1.6, and 1.8 kb, with the 1.6- and 1.8-kb fragments appearing only during the last half of the study period. Examination of linkages among the different resistance genes showed a variety of positive and negative associations. Association analysis of isolates divided into two groups, those isolated between 1978 and 1989 and those isolated between 1990 and 2000, revealed the appearance of new positive resistance gene associations. Our genotypic resistance analyses of ETEC isolates from pigs indicate that many of the antibiotic resistance genes behind phenotypic resistance are not static but, rather, are in a state of flux driven by various selection forces such as the use of specific antimicrobials.

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.

The toxicity and toxicogenomics of selected anatase and rutile nanoparticles (NP) and bulk titanium dioxide (TiO2) particles were evaluated in the soil nematode Caenorhabditis elegans. Results indicated that bulk or nano-TiO2 particles were slightly toxic to soil nematode C. elegans, as measured by reproduction EC50 values ranging from 4 to 32 mg/L. Whole-genome microarray results indicated that the regulation of glutathione-S-transferase gst-3, cytochrome P450 cypp33-c11, stress resistance regulator scl-1, oxidoreductase wah-1 and embryonic development pod-2 genes were significantly affected by nano-sized and bulk-TiO2 particles. More specifically, it was determined that anatase particles exerted a greater effect on metabolic pathways, whereas rutile particles had a greater effect on developmental processes. The up-regulation of the pod-2 gene corroborated the phenotypic effect observed in the reproduction test. Our results demonstrated that C. elegans is a good genomic model for nano-TiO2 toxicity assessment.

Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants.

Wastewater discharges may increase the populations of pathogens, including Escherichia coli , and of antimicrobial-resistant strains in receiving waters. This study investigated the impact of UV and peracetic acid (PAA) disinfection on the prevalence of virulence and antimicrobial resistance genes in uropathogenic Escherichia coli (UPEC), the most abundant E. coli pathotype in municipal wastewaters. Laboratory disinfection experiments were conducted on wastewater treated by physicochemical, activated sludge, or biofiltration processes; 1,766 E. coli isolates were obtained for the evaluation. The target disinfection level was 200 CFU/100 ml, resulting in UV and PAA doses of 7 to 30 mJ/cm 2 and 0.9 to 2.0 mg/liter, respectively. The proportions of UPECs were reduced in all samples after disinfection, with an average reduction by UV of 55% (range, 22% to 80%) and by PAA of 52% (range, 11% to 100%). Analysis of urovirulence genes revealed that the decline in the UPEC populations was not associated with any particular virulence factor. A positive association was found between the occurrence of urovirulence and antimicrobial resistance genes (ARGs). However, the changes in the prevalence of ARGs in potential UPECs were different following disinfection, i.e., UV appears to have had no effect, while PAA significantly reduced the ARG levels. Thus, this study showed that both UV and PAA disinfections reduced the proportion of UPECs and that PAA disinfection also reduced the proportion of antimicrobial resistance gene-carrying UPEC pathotypes in municipal wastewaters.

Enterohaemorrhagic Escherichia coli (EHEC) are responsible for outbreaks of food- and water-borne illness. The bovine gastrointestinal tract (GIT) is thought to be the principle reservoir of EHEC. Knowledge of the nutrients essential for EHEC growth and survival in the bovine intestine may help in developing strategies to limit their shedding in bovine faeces thus reducing the risk of human illnesses. To identify specific metabolic pathways induced in the animal GIT, the transcriptome profiles of EHEC O157:H7 EDL933 during incubation in bovine small intestine contents (BSIC) and minimal medium supplemented with glucose were compared. The transcriptome analysis revealed that genes responsible for the assimilation of ethanolamine, urea, agmatine and amino acids (Asp, Thr, Gly, Ser and Trp) were strongly up-regulated suggesting that these compounds are the main nitrogen sources for EHEC in BSIC. A central role for the gluconeogenesis pathway and assimilation of gluconeogenic substrates was also pinpointed in EHEC incubated in BSIC. Our results suggested that three amino acids (Asp, Ser and Trp), glycerol, glycerol 3-phosphate, L-lactate and C4-dicarboxylates are important carbon sources for EHEC in BSIC. The ability to use gluconeogenic substrates as nitrogen sources (amino acids) and/or carbon sources (amino acids, glycerol and lactate) may provide a growth advantage to the bacteria in intestinal fluids. Accordingly, aspartate (2.4 mM), serine (1.9 mM), glycerol (5.8 mM) and lactate (3.6 mM) were present in BSIC and may represent the main gluconeogenic substrates potentially used by EHEC. A double mutant of E. coli EDL933 defective for phosphoenolpyruvate synthase (PpsA) and phosphoenolpyruvate carboxykinase (PckA), unable to utilize tricarboxylic acid (TCA) intermediates was constructed. Growth competition experiments between EHEC EDL933 and the isogenic mutant strain in BSIC clearly showed a significant competitive growth advantage of the wild-type strain further illustrating the importance of the gluconeogenesis pathway in maintaining EHEC in the bovine GIT. © 2014 Bertin et al.