Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species.

AIM: A single-tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp. METHODS AND RESULTS: Primer pairs for each target species (except A. skirrowii) and mPCR protocol were newly designed and optimized using variable regions of housekeeping including cpn60, gyrA, gyrB and rpoB genes. The accuracy and specificity of the mPCR assay was assessed using DNA templates from six targets and 11 other Arcobacter spp. as well as 50 other bacterial reference species and strains. Tests on the DNA templates of target Arcobacter spp. were appropriately identified, whereas all 61 other DNA templates from other bacterial species and strains were not amplified. Sensitivity and specificity of the mPCR assay was 10 pg µl-1 of DNA concentration per target species. The optimized assay was further evaluated, validated and compared with other mPCR assays by testing Arcobacter cultures isolated from various faecal and water sources. CONCLUSIONS: Study results confirm that the newly developed mPCR assay is rapid, accurate, reliable, simple, and valuable for the simultaneous detection and routine diagnosis of six human- and animal-associated Arcobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The new mPCR assay is useful not only for pure but also mixed cultures. Moreover, it has the ability to rapidly detect six species which enhances the value of this technology for aetiological and epidemiological studies.

Comparison of commercial DNA extraction kits and quantitative PCR systems for better sensitivity in detecting the causative agent of paratuberculosis in dairy cow fecal samples.

Mycobacterium avium ssp. paratuberculosis (MAP) causes ruminant paratuberculosis (Johne's disease) worldwide. Oral-fecal contamination is the most important mode of transmission of paratuberculosis, so eradicating MAP-shedding animals could prevent disease propagation. Fecal culture, a well-known method for MAP diagnosis, requires costly specialized media and a long incubation time that sometimes ends in disappointing bacterial contamination. To facilitate the efforts of control programs, we evaluated the performance of direct fecal quantitative PCR (qPCR) assays for their sensitivity and robustness for MAP detection. Commercial kits use different strategies for extracting DNA, combined with qPCR systems, to detect the presence of MAP in fecal samples. In this study, we compared the sensitivity of 3 commercially available DNA extraction kits (A, B, and C) combined with 2 qPCR systems (T and V) for the detection of MAP in infectious cows. A total of 49 dairy cows from 5 herds were sampled twice a year for 3 yr and diagnosed using fecal culture and ELISA. Eight replicates of their fecal samples from the first sampling were tested using each DNA extraction method and qPCR detection system. Although all 3 of the commercial DNA extraction kits have been previously described as very efficient for the diagnosis of paratuberculosis, kit B provided the highest sensitivity. Indeed, 89% of the cows declared positive for paratuberculosis by both fecal culture and ELISA were identified with kit B, whereas only 23 and 43% of the cows were identified with kits A and C, respectively. Interestingly, kit B was able to detect some low-MAP shedders. The qPCR detection system also played a critical role: system T yielded qPCR with the highest sensitivity. The results of this study suggest that DNA extraction kit B combined with detection system T provides the best amplification of MAP DNA from fecal samples with the highest sensitivity and specificity. Although 1 DNA extraction and qPCR analysis should be adequate to confirm that an animal with diarrhea or other signs of paratuberculosis is positive, detecting low shedders at the highest sensitivity should include repetitive testing. This study demonstrates the importance of repetitions using the most appropriate method for extracting DNA from fecal samples, combined with a compatible qPCR system for identifying MAP-shedding animals.

Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model.

PURPOSE: To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates. METHODS: IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines. RESULTS: IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs). CONCLUSIONS: Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.

Using AnnAGNPS to Predict the Effects of Tile Drainage Control on Nutrient and Sediment Loads for a River Basin.

Controlled tile drainage (CTD) can reduce pollutant loading. The Annualized Agricultural Nonpoint Source model (AnnAGNPS version 5.2) was used to examine changes in growing season discharge, sediment, nitrogen, and phosphorus loads due to CTD for a ∼3900-km agriculturally dominated river basin in Ontario, Canada. Two tile drain depth scenarios were examined in detail to mimic tile drainage control for flat cropland: 600 mm depth (CTD) and 200 mm (CTD) depth below surface. Summed for five growing seasons (CTD), direct runoff, total N, and dissolved N were reduced by 6.6, 3.5, and 13.7%, respectively. However, five seasons of summed total P, dissolved P, and total suspended solid loads increased as a result of CTD by 0.96, 1.6, and 0.23%. The AnnAGNPS results were compared with mass fluxes observed from paired experimental watersheds (250, 470 ha) in the river basin. The "test" experimental watershed was dominated by CTD and the "reference" watershed by free drainage. Notwithstanding environmental/land use differences between the watersheds and basin, comparisons of seasonal observed and predicted discharge reductions were comparable in 100% of respective cases. Nutrient load comparisons were more consistent for dissolved, relative to particulate water quality endpoints. For one season under corn crop production, AnnAGNPS predicted a 55% decrease (CTD) in dissolved N from the basin. AnnAGNPS v. 5.2 treats P transport from a surface pool perspective, which is appropriate for many systems. However, for assessment of tile drainage management practices for relatively flat tile-dominated systems, AnnAGNPS may benefit from consideration of P and particulate transport in the subsurface.

Long-Term Observations of Nitrogen and Phosphorus Export in Paired-Agricultural Watersheds under Controlled and Conventional Tile Drainage.

Controlled tile drainage (CTD) regulates water and nutrient export from tile drainage systems. Observations of the effects of CTD imposed en masse at watershed scales are needed to determine the effect on downstream receptors. A paired-watershed approach was used to evaluate the effect of field-to-field CTD at the watershed scale on fluxes and flow-weighted mean concentrations (FWMCs) of N and P during multiple growing seasons. One watershed (467-ha catchment area) was under CTD management (treatment [CTD] watershed); the other (250-ha catchment area) had freely draining or uncontrolled tile drainage (UCTD) (reference [UCTD] watershed). The paired agricultural watersheds are located in eastern Ontario, Canada. Analysis of covariance and paired tests were used to assess daily fluxes and FWMCs during a calibration period when CTD intervention on the treatment watershed was minimal (2005-2006, when only 4-10% of the tile-drained area was under CTD) and a treatment period when the treatment (CTD) watershed had prolific CTD intervention (2007-2011 when 82% of tile drained fields were controlled, occupying >70% of catchment area). Significant linear regression slope changes assessed using ANCOVA ( ≤ 0.1) for daily fluxes from upstream and downstream monitoring sites pooled by calibration and treatment period were -0.06 and -0.20 (stream water) (negative values represent flux declines in CTD watershed), -0.59 and -0.77 (NH-N), -0.14 and -0.15 (NO-N), -1.77 and -2.10 (dissolved reactive P), and -0.28 and 0.45 (total P). Total P results for one site comparison contrasted with other findings likely due to unknown in-stream processes affecting total P loading, not efficacy of CTD. The FWMC results were mixed and inconclusive but suggest physical abatement by CTD is the means by which nutrient fluxes are predominantly reduced at these scales. Overall, our study results indicate that CTD is an effective practice for reducing watershed scale fluxes of stream water, N, and P during the growing season.

Rainfall-induced runoff from exposed streambed sediments: an important source of water pollution.

When surface water levels decline, exposed streambed sediments can be mobilized and washed into the water course when subjected to erosive rainfall. In this study, rainfall simulations were conducted over exposed sediments along stream banks at four distinct locations in an agriculturally dominated river basin with the objective of quantifying the potential for contaminant loading from these often overlooked runoff source areas. At each location, simulations were performed at three different sites. Nitrogen, phosphorus, sediment, fecal indicator bacteria, pathogenic bacteria, and microbial source tracking (MST) markers were examined in both prerainfall sediments and rainfall-induced runoff water. Runoff generation and sediment mobilization occurred quickly (10-150 s) after rainfall initiation. Temporal trends in runoff concentrations were highly variable within and between locations. Total runoff event loads were considered large for many pollutants considered. For instance, the maximum observed total phosphorus runoff load was on the order of 1.5 kg ha. Results also demonstrate that runoff from exposed sediments can be a source of pathogenic bacteria. spp. and spp. were present in runoff from one and three locations, respectively. Ruminant MST markers were also present in runoff from two locations, one of which hosted pasturing cattle with stream access. Overall, this study demonstrated that rainfall-induced runoff from exposed streambed sediments can be an important source of surface water pollution.

Comprehensive nitrogen budgets for controlled tile drainage fields in eastern ontario, Canada.

Excessive N loading from subsurface tile drainage has been linked to water quality degradation. Controlled tile drainage (CTD) has the potential to reduce N losses via tile drainage and boost crop yields. While CTD can reduce N loss from tile drainage, it may increase losses through other pathways. A multiple-year field-scale accounting of major N inputs and outputs during the cropping season was conducted on freely drained and controlled tile drained agricultural fields under corn ( L.)-soybean [ (L.) Merr.] production systems in eastern Ontario, Canada. Greater predicted gaseous N emissions for corn and soybean and greater observed lateral seepage N losses were observed for corn and soybean fields under CTD relative to free-draining fields. However, observed N losses from tile were significantly lower for CTD fields, in relation to freely drained fields. Changes in residual soil N were essentially equivalent between drainage treatments, while mass balance residual terms were systematically negative (slightly more so for CTD). Increases in plant N uptake associated with CTD were observed, probably resulting in higher grain yields for corn and soybean. This study illustrates the benefits of CTD in decreasing subsurface tile drainage N losses and boosting crop yields, while demonstrating the potential for CTD to increase N losses via other pathways related to gaseous emissions and groundwater seepage.

Measuring and modeling the effects of drainage water management on soil greenhouse gas fluxes from corn and soybean fields.

Controlled tile drainage can boost crop yields and improve water quality, but it also has the potential to increase GHG emissions. This study compared in-situ chamber-based measures of soil CH4, N2O, and CO2 fluxes for silt loam soil under corn and soybean cropping with conventional tile drainage (UTD) and controlled tile drainage (CTD). A semi-empirical model (NEMIS-NOE) was also used to predict soil N2O fluxes from soils using observed soil data. Observed N2O and CH4 fluxes between UTD and CTD fields during the farming season were not significantly different at 0.05 level. Soils were primarily a sink for CH4 but in some cases a source (sources were associated exclusively with CTD). The average N2O fluxes measured ranged between 0.003 and 0.028 kg N ha(-1) day(-1). There were some significantly higher (p ≤ 0.05) CO2 fluxes associated with CTD relative to UTD during some years of study. Correlation analyses indicated that the shallower the water table, the greater the CO2 fluxes. Higher corn plant C for CTD tended to offset estimated higher CTD CO2 C losses via soil respiration by ∼100-300 kg C ha(-1). There were good fits between observed and predicted (NEMIS-NOE) N2O fluxes for corn (R(2) = 0.70) and soybean (R(2) = 0.53). Predicted N2O fluxes were higher for CTD for approximately 70% of the paired-field study periods suggesting that soil physical factors, such as water-filled pore space, imposed by CTD have potentially strong impacts on net N fluxes. Model predictions of daily cumulative N2O fluxes for the agronomically-active study period for corn-CTD and corn-UTD, as a percentage of total N fertilizer applied, were 3.1% and 2.6%, respectively. For predicted N2O fluxes on basis of yield units, indices were 0.0005 and 0.0004 (kg N kg(-1) crop grain yield) for CTD and UTD corn fields, respectively, and 0.0011 and 0.0005 for CTD and UTD soybean fields, respectively.

Impact of riparian zone protection from cattle on nutrient, bacteria, F-coliphage, and loading of an intermittent stream.

This 5-yr study compared, via an upstream-downstream experimental design, nutrient and microbial water quality of an intermittent stream running through a small pasture (∼2.5 animals ha) where cattle are restricted from the riparian zone (restricted cattle access [RCA]) and where cattle have unrestricted access to the stream (unrestricted cattle access [URCA]). Fencing in the RCA excluded pasturing cattle to within ∼3 to 5 m of the stream. Approximately 88% (26/32) of all comparisons of mean contaminant load reduction for lower, higher, and all stream flow conditions during the 5-yr study indicated net contaminant load reductions in the RCA; for the URCA, this percentage was 38% (12/32). For all flow conditions, mean percent load reductions in the RCA for nutrients and bacteria plus F-coliphage were 24 and 23%, respectively. These respective percentages for the URCA were -9 and -57% (positive values are reductions; negative values are increases). However, potentially as a result of protected wildlife habitat in the RCA, the mean percent load reduction for for "all flow" was -321% for the RCA and 60% for the URCA; for , these respective percentages were -209% (RCA) and 73% (URCA). For "all flow" situations, mean load reductions for the RCA were significantly greater ( < 0.1) than those from the URCA for NH-N, dissolved reactive phosphorus (DRP), total coliform, , and . For "high flow" situations, mean load reductions were significantly greater for the RCA for DRP, total coliform, and . For "low flow" conditions, significantly greater mean load reductions were in favor of the RCA for DRP, total P, total coliforms, fecal coliforms, , and . In no case were mean pollutant loads in the URCA significantly higher than RCA pollutant loads. Restricting pasturing livestock to within 3 to 5 m of intermittent streams can improve water quality; however, water quality impairment can occur if livestock have unrestricted access to a stream.

Investigation of an Escherichia coli environmental benchmark for waterborne pathogens in agricultural watersheds in Canada.

Canada's National Agri-Environmental Standards Initiative sought to develop an environmental benchmark for low-level waterborne pathogen occurrence in agricultural watersheds. A field study collected 902 water samples from 27 sites in four intensive agricultural watersheds across Canada from 2005 to 2007. Four of the sites were selected as reference sites away from livestock and human fecal pollution sources in each watershed. Water samples were analyzed for Campylobacter spp., Salmonella spp., Escherichia coli O157:H7, Cryptosporidium spp., Giardia spp., and the water quality indicator E. coli. The annual mean number of pathogen species was higher at agricultural sites (1.54 ± 0.07 species per water sample) than at reference sites (0.75 ± 0.14 species per water sample). The annual mean concentration of E. coli was also higher at agricultural sites (491 ± 96 colony-forming units [cfu] 100 mL(-1)) than at reference sites (53 ± 18 cfu 100 mL(-1)). The feasibility of adopting existing E. coli water quality guideline values as an environmental benchmark was assessed, but waterborne pathogens were detected at agricultural sites in 80% of water samples with low E. coli concentrations (<100 cfu 100 mL(-1)). Instead, an approach was developed based on using the natural background occurrence of pathogens at reference sites in agricultural watersheds to derive provisional environmental benchmarks for pathogens at agricultural sites. The environmental benchmarks that were derived were found to represent E. coli values lower than geometric mean values typically found in recreational water quality guidelines. Additional research is needed to investigate environmental benchmarks for waterborne pathogens within the context of the "One World, One Health" perspective for protecting human, domestic animal, and wildlife health.

Use of glass transitions in carbohydrate excipient design for lyophilized protein formulations.

This work describes an effort to apply methods from process systems engineering to a pharmaceutical product design problem, with a novel application of statistical approaches to comparing solutions. A computational molecular design framework was employed to design carbohydrate molecules with high glass transition temperatures and low water content in the maximally freeze-concentrated matrix, with the objective of stabilizing lyophilized protein formulations. Quantitative structure-property relationships were developed for glass transition temperature of the anhydrous solute, glass transition temperature of the maximally concentrated solute, melting point of ice and Gordon-Taylor constant for carbohydrates. An optimization problem was formulated to design an excipient with optimal property values. Use of a stochastic optimization algorithm, Tabu search, provided several carbohydrate excipient candidates with statistically similar property values, as indicated by prediction intervals calculated for each property.

Pathogenic and multidrug-resistant Escherichia fergusonii from broiler chicken.

An Escherichia spp. isolate, ECD-227, was previously identified from the broiler chicken as a phylogenetically divergent and multidrug-resistant Escherichia coli possessing numerous virulence genes. In this study, whole genome sequencing and comparative genome analysis was used to further characterize this isolate. The presence of known and putative antibiotic resistance and virulence open reading frames were determined by comparison to pathogenic (E. coli O157:H7 TW14359, APEC O1:K1:H7, and UPEC UTI89) and nonpathogenic species (E. coli K-12 MG1655 and Escherichia fergusonii ATCC 35469). The assembled genome size of 4.87 Mb was sequenced to 18-fold depth of coverage and predicted to contain 4,376 open reading frames. Phylogenetic analysis of 537 open reading frames present across 110 enteric bacterial species identifies ECD-227 to be E. fergusonii. The genome of ECD-227 contains 5 plasmids showing similarity to known E. coli and Salmonella enterica plasmids. The presence of virulence and antibiotic resistance genes were identified and localized to the chromosome and plasmids. The mutation in gyrA (S83L) involved in fluoroquinolone resistance was identified. The Salmonella-like plasmids harbor antibiotic resistance genes on a class I integron (aadA, qacEΔ-sul1, aac3-VI, and sulI) as well as numerous virulence genes (iucABCD, sitABCD, cib, traT). In addition to the genome analysis, the virulence of ECD-227 was evaluated in a 1-d-old chick model. In the virulence assay, ECD-227 was found to induce 18 to 30% mortality in 1-d-old chicks after 24 h and 48 h of infection, respectively. This study documents an avian multidrug-resistant and virulent E. fergusonii. The existence of several resistance genes to multiple classes of antibiotics indicates that infection caused by ECD-227 would be difficult to treat using antimicrobials currently available for poultry.

Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry.

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.

Distribution of selected virulence genes and antibiotic resistance in Enterococcus species isolated from the South Nation River drainage basin, Ontario, Canada.

AIMS: Isolate and characterize water enterococci from the South Nation River drainage basin, an area dominated by agriculture. METHODS AND RESULTS: A total of 1558 enterococci were isolated from 204 water samples from the South Nation River obtained over a 3-year period. PCR was used to identify isolates to the species level and characterize them for carriage of 12 virulence determinants. Antibiotic resistance was evaluated phenotypically. Enterococcus faecalis (36·4%), Enterococcus faecium (9·3%) and Enterococcus durans (8·5%) were the major enterococci species isolated. Enterococci carrying more than two virulence determinants were more frequently detected in the summer (59·6%) than in other seasons (≤ 37·6%). Very few isolates (≤ 2·0%) were resistant to category I antibiotics ciprofloxacin and vancomycin. CONCLUSIONS: Comparison of major water enterococci species with major faecal enterococci species obtained from various host groups (human, domesticated mammals and birds, wildlife) in this drainage basin suggest that water enterococci may have varied faecal origins. The low level of antibiotic resistance among enterococci suggests that dispersion of antibiotic resistance via waterborne enterococci in this watershed is not significant. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study suggests that water enterococci in the SNR have a faecal origin and that their potential impact on public health regarding antibiotic resistance and virulence determinants is minimal.

Continuous feeding of antimicrobial growth promoters to commercial swine during the growing/finishing phase does not modify faecal community erythromycin resistance or community structure.

AIMS: To investigate the effect of continuous feeding of antimicrobial growth promoters (tylosin or virginiamycin) on the swine faecal community. METHODS AND RESULTS: The study consisted of two separate on-farm feeding trials. Swine were fed rations containing tylosin (44 or 88 mg kg(-1) of feed) or virginiamycin (11 or 22 mg kg(-1) of feed) continuously over the growing/finishing phases. The temporal impact of continuous antimicrobial feeding on the faecal community was assessed and compared to nondosed control animals through anaerobic cultivation, the analysis of community 16S rRNA gene libraries and faecal volatile fatty acid content. Feeding either antimicrobial had no detectable effect on the faecal community. CONCLUSIONS: Erythromycin methylase genes encoding resistance to the macrolide-lincosamide-streptogramin B (MLS(B) ) antimicrobials are present at a high level within the faecal community of intensively raised swine. Continuous antimicrobial feeding over the entire growing/finishing phase had no effect on community erm-methylase gene copy numbers or faecal community structure. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial growth promoters are believed to function by altering gut bacterial communities. However, widespread MLS(B) resistance within the faecal community of intensively raised swine likely negates any potential effects that these antimicrobials might have on altering the faecal community. These findings suggest that if AGP-mediated alterations to gut communities are an important mechanism for growth promotion, it is unlikely that these would be associated with the colonic community.

Longitudinal characterization of resistant Escherichia coli in fecal deposits from cattle fed subtherapeutic levels of antimicrobials.

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp(r)) and tetracycline-resistant (Tet(r)) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp(r) and Tet(r) E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet(r) E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet(r) and Amp(r) E. coli. Except for Amp(r) E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet(r) E. coli numbers were detected in A44 and AS700 deposits [5.9 log(10) CFU (g dry matter)(-1) and 5.4 log(10) CFU (g dry matter)(-1), respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla(TEM1), tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.

Comparative microscale analysis of the effects of triclosan and triclocarban on the structure and function of river biofilm communities.

The broad spectrum antimicrobials triclosan (TCS) and triclocarban (TCC) are commonly detected in the environment. However, there is very limited understanding of the aquatic ecological implications of these agents. During this study, river biofilms were cultivated using 10 microg l(-1) of TCS or TCC and the equivalent in nutrients (carbon, nitrogen) over a developmental period of 8 weeks. Confocal laser microscopy showed that the biofilm communities developing under the influence of TCS and TCC had community architecture and composition different from either control or nutrient exposed communities. Microscale analyses of biofilm community structure indicated a significant reduction in algal biomass (p<0.05) as a result of exposure to either TCS or TCC. Thymidine incorporation did not detect significant differences between control and treated communities. The use of carbon utilization assays based on growth indicated that, in general, TCS and TCC suppressed utilization. The community was altered from one dominated by autotrophic processes to one dominated by heterotrophic processes. Both TCS and TCC treatments resulted in significant (p<0.05) alterations in the composition of the EPS matrix of the communities, suggesting significant changes in community composition. Denaturing gradient gel electrophoresis and PCA-ANOSIM analyses indicated a significant change occurred in the bacterial community as a consequence of TCS treatments. Enumeration of micrometazoa and protozoa revealed an increase in micrometazoan numbers over control values, whereas no clear impact on protozoa was detected in any treatment. This study indicated significant effects of 10 microg l(-1) TCS and TCC on microbial community composition, algal biomass, architecture and activity.

Nitrogen, phosphorus, and bacteria tile and groundwater quality following direct injection of dewatered municipal biosolids into soil.

Application of municipal biosolids (sewage) to agricultural land is a common practice to improve soil physical quality and fertility. The chosen method of land application can have a strong impact on the extent of adjacent water contamination by nutrients and bacteria. Dewatered municipal biosolids (DMB) were applied to silt-clay loam experimental field plots in Ontario, Canada using two application methods: (i) surface spreading followed by shallow incorporation (SS) and (ii) a newly developed implement that directly injects DMB into the topsoil (DI). The objective of this study was to compare N, P, and bacteria quality of tile drainage and shallow groundwater associated with each land application technique. There were no significant differences (P > 0.05) in N, P, and bacteria tile mass loads among the application treatments for time periods <100 d postapplication, when the greatest peak loads and peak tile water concentrations were observed. Both land application treatments caused groundwater Escherichia coli contamination to at least 1.2 m depth below surface after the first postapplication rainfall event, and NO(3)-N contamination to at least 2.0 m depth below surface. The DI treatment did, however, have significantly (P < 0.05) higher tile mass loads of total Kjeldahl N (TKN), total phosphorus (TP), E. coli, Enterococci, and Clostridium perfringens relative to the SS treatment for time periods >100 d postapplication. Nevertheless, relative to tile effluent data collected <100 d postapplication (no application treatment differences), peak loads, and concentrations during this time were, overall, considerably lower for both treatments. This finding, along with no significant differences in N, P, and bacteria groundwater concentrations among the application treatments, and that the direct injection technique could potentially reduce vector attraction problems and odor, suggests that the direct injection technique should be considered a dewatered municipal biosolid land application option.

A computational molecular design framework for crosslinked polymer networks.

Crosslinked polymers are important in a very wide range of applications including dental restorative materials. However, currently used polymeric materials experience limited durability in the clinical oral environment. Researchers in the dental polymer field have generally used a time-consuming experimental trial-and-error approach to the design of new materials. The application of computational molecular design (CMD) to crosslinked polymer networks has the potential to facilitate development of improved polymethacrylate dental materials. CMD uses quantitative structure property relations (QSPRs) and optimization techniques to design molecules possessing desired properties. This paper describes a mathematical framework which provides tools necessary for the application of CMD to crosslinked polymer systems. The novel parts of the system include the data structures used, which allow for simple calculation of structural descriptors, and the formulation of the optimization problem. A heuristic optimization method, Tabu Search, is used to determine candidate monomers. Use of a heuristic optimization algorithm makes the system more independent of the types of QSPRs used, and more efficient when applied to combinatorial problems. A software package has been created which provides polymer researchers access to the design framework. A complete example of the methodology is provided for polymethacrylate dental materials.