Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry.

In vitro sensitivity of poultry Brachyspira intermedia isolates to essential oil components and in vivo reduction of Brachyspira intermedia in rearing pullets with cinnamaldehyde feed supplementation.

Cecal enteritis due to Brachyspira infections tends to be chronic in laying hens. Limited availability of antimicrobial drugs for use in laying hens emphasizes the need for alternative control measures. A broth microdilution method was used to determine the antimicrobial susceptibility of 20 Brachyspira intermedia field isolates from laying hen flocks to components of essential oils (EO). Minimal inhibitory concentration (MIC) distributions, obtained for 8 EO components, were all monomodal. Cinnamaldehyde had the lowest MIC values (40 to 80 mg/L), followed by nerolidol, capsaicin, carvacrol, and thymol (80 to 320 mg/L), eugenol (160 to 640 mg/L), and linalool (320 to 1,280 mg/L). The MIC ranges of piperine were mostly above the test range of 1,280 mg/L. In an in vivo experiment, coated trans-cinnamaldehyde was supplemented to the feed of rearing pullets. A completely randomized experimental design with 4 treatments and 3 replicates each (replicate = group of seven 1-d-old laying hen chickens) was applied. The negative and positive controls received a conventional feed during the whole trial. The positive controls were orally inoculated on 3 consecutive days (d 22, 23, and 24) with 1 mL of 1.0 × 10(8) cfu/mL of a B. intermedia field isolate. Two treatment groups (preventive and curative), identically inoculated, received the coated trans-cinnamaldehyde-supplemented feed (500 mg/kg of trans-cinnamaldehyde), the preventive group from d 1, the curative from d 25. On d 32, ceca were collected for bacteriologic Brachyspira enumeration. The mean enumeration of Brachyspira cells was decreased (P < 0.05) in the curative treated group versus the positive control group. The in vitro results of the present study demonstrate the potential of EO components as antimicrobials against poultry Brachyspira isolates, including isolates with acquired resistance for classic antimicrobial drugs. Reduction of Brachyspira colonization in young pullets was obtained, in a curative way, in an in vivo study using feed supplemented with coated trans-cinnamaldehyde. Further studies are necessary to investigate the mode of action of the coated trans-cinnamaldehyde in reducing Brachyspira colonization of the ceca.

Effects of sorption, sulphate reduction, and Phragmites australis on the removal of heavy metals in subsurface flow constructed wetland microcosms.

The removal of Co, Ni, Cu and Zn from synthetic industrial wastewater was studied in subsurface flow constructed wetland microcosms filled with gravel or a gravel/straw mixture. Half of the microcosms were planted with Phragmites australis and half were left unplanted. All microcosms received low-strength wastewater (1 mg L(-1) of Co, Ni, and Zn, 0.5 mg L(-1) Cu, 2,000mg L(-1) SO4) during seven 14-day incubation batches. The pore water was regularly monitored at two depths for heavy metals, sulphate, organic carbon and redox potential. Sorption properties of gravel and straw were assessed in a separate experiment. A second series of seven incubation batches with high-strength wastewater (10 mg L(-1) of each metal, 2,000 mg L(-1) SO4) was then applied to saturate the substrate. Glucose was added to the gravel microcosms together with the high-strength wastewater. Sorption processes were responsible for metal removal during start-up, with the highest removal efficiencies in the gravel microcosms. The lower initial efficiencies in the gravel/straw microcosms were presumably caused by the decomposition of straw. However, after establishment of anaerobic conditions (Eh approximately -200 mV), precipitation as metal sulphides provided an additional removal pathway in the gravel/straw microcosms. The addition of glucose to gravel microcosms enhanced sulphate reduction and metal removal, although Phragmites australis negatively affected these processes in the top-layer of all microcosms.

Accumulation of metals in a horizontal subsurface flow constructed wetland treating domestic wastewater in Flanders, Belgium.

This study assessed the accumulation of metals in a horizontal subsurface flow constructed wetland treating domestic wastewater of 350 PE after three years of operation. Metal concentrations in the influent wastewater, effluent, sediment, leaves, stems, and belowground biomass of Phragmites australis were analysed. Spatial variations were assessed by sampling at increasing distance from the inlet and at different positions across the width of the reed bed. All metals except Fe and Mn were efficiently removed in the CW, total metal concentrations in the effluent complied with basic environmental quality standards for surface water, and dissolved metal concentrations were often lower than analytical detection limits. Removal efficiencies varied between 49% for Ni and 93% for Al. Export of dissolved Mn and particulate Fe occurred, probably related to redox conditions in the sediment. After 3 years of operation, the sediment in the inlet area was significantly contaminated with Zn, Cu, and Cd, whereas Pb could form a contamination problem within the near future. The Cr and Ni levels in the sediment were low throughout the entire reed bed. At this stage of operation, the contamination problem was still situated within the inlet area and metal concentrations in the sediment decreased towards background values further along the treatment path. An exponential decrease of the metal mass in the sediment and belowground biomass was seen for all metals except Mn. Contrary to the other metals, Mn concentrations in the sediment increased with distance. For all metals, less than 2% of the mass removed from the wastewater after passage through the reed bed is accumulated in the aboveground reed biomass. The sediment acts as the primary sink for metals.

Application of automated measurement stations for continuous water quality monitoring of the Dender river in Flanders, Belgium.

During the summer of 1999, two automated water quality measurement stations were installed along the Dender river in Belgium. The variables dissolved oxygen, temperature, conductivity, pH, rain-intensity, flow and solar radiation were measured continuously. In this paper these on-line measurement series are presented and interpreted using also additional measurements and ecological expert-knowledge. The purpose was to demonstrate the variability in time and space of the aquatic processes and the consequences of conducting and interpreting discrete measurements for river quality assessment and management. The large fluctuations of the data illustrated the importance of continuous measurements for the complete description and modelling of the biological processes in the river.

Impact of operational maintenance on the asset life of storm reed beds.

This paper reviews the operation of storm reed beds to determine whether the current system of operational maintenance is contributing to premature process failures and if not, to identify other factors of importance. Twelve storm reed beds of the horizontal subsurface flow type, at seven locations in the South Warwickshire area of the United Kingdom, were surveyed. Each survey consisted of a site visit, an interview with the operators in charge and an assessment of the treatment performance based on routine monitoring data. Although some sites suffered from varying degrees of sludge accumulation, surface blinding and/or weed growth, all effluent concentrations remained far below the consent levels. Thorough operational maintenance on a reed bed is proven to be important for the asset life. However, there are other factors or features of a reed bed that play a more pivotal role in premature process failure such as the lack of pretreatment and a premature operation of the storm overflow.

Impact of land-use on macroinvertebrate communities in the Zwalm river basin.

For determining the impact of land-use on the aquatic ecosystem in a river basin, a Geographic Information System (GIS) has been used to integrate the main impact factors at different scales. The headwaters and streams of the Zwalm River basin (Flanders, Belgium), which is part of the Scheldt river basin, were selected as study site. A river ecological database was developed based on physical-chemical and biological monitoring. The research focussed on three zones within this Zwalm River basin, each characterized by different land-uses and by consequence different types of pollution, mainly of diffuse origin. The impact was measured by means of the biological diversity, the Belgian Biotic Index and a key macroinvertebrate taxon. For measuring, assessing and managing the impact of land-use on biological communities, an integrated and multidisciplinary approach will be necessary.

Development of short and long-term management options for bergelenput to avoid fish kills caused by algal blooms.

Numerous stagnant waters in Flanders are characterized by high nutrient concentrations, leading to severe algal blooms and under critical conditions to fish mortality. In this context, a eutrophicated pond was analysed hydrobiologically during one month in order to develop a management plan for the coming years to restore its ecological stability. Bergelenput (Wevelgem), a former sand pit, is now a nature reserve, currently also used for recreational fishing. During the last decade, incidental fish kills have occurred. The main cause is thought to be nutrient enrichment, enhancing algal blooms. The most probable source of these nutrients is fertiliser runoff from the surrounding fields characterised by intensive agricultural activities. Two possible causes of these fish kills, both associated with algal blooms, were considered during this preliminary study. The first was oxygen depletion of the water caused by algal respiration during the night. The second was related to the presence of Microcystis aeruginosa, a cyanobacterium known to be toxic sometimes and hence responsible for fish kills. Short-term and long-term management options are being developed within the context of this research to rehabilitate the ecosystem.

Development of river ecosystem models for Flemish watercourses: case studies in the Zwalm river basin.

Only recently, modelling has been accepted as an interesting and powerful tool to support river quality assessment and management. The 'River Invertebrate Prediction and Classification System' (RIVPACS), based on statistical modelling, was one of the first and best known systems in this context. RIVPACS was developed to classify macroinvertebrate community types and to predict the fauna expected to occur in different types of watercourses, based on a small number of environmental variables. The prediction is essentially a static 'target' of the fauna to be expected at a site with stated environmental features, in the absence of environmental stress. Therefore this system is rather limited to apply in river assessment and management. Models that offer a prediction of faunal responses to changes in environmental features (e.g. changes in discharge regime, dissolved oxygen level, ...) would be of considerable value for river management. In this context, models based on classification trees, artificial neural networks and fuzzy logic were developed and applied to predict macro-invertebrate communities in the Zwalm river basin located in Flanders, Belgium. Structural characteristics (meandering, substrate type, flow velocity, ...) and physical-chemical variables (dissolved oxygen, pH, ...) were used as inputs to predict the presence or absence of macroinvertebrate taxa in the headwaters and brooks of the Zwalm river basin. In total, data from 60 measurement sites were available. Reliability and particular strengths and weaknesses of these techniques were compared and evaluated. Classification trees performed in general well to predict the absence or presence of the different macroinvertebrate taxa and allowed also to deduct general relations from the dataset. Models based on artificial neural networks (ANNS) were also good in predicting the macroinvertebrate communities at the different sites. Sensitivity analyses related to ANNs allowed to study the impact of the input variables on the presence or absence of macroinvertebrate taxa and to determine the major variables that affect the ecosystem quality and should be taken under direct consideration in the management of river basins. Performance of the fuzzy logic models was significantly related to the methods that were used to set up the membership functions and the reliability of the information that was available. Fuzzy logic did not perform as well as the other two techniques with regard to short term predictions. Fuzzy logic appeared to be better and more robust for long term predictions, because of the easy and pragmatic integration of general expert knowledge and data derived rules in the transparent inference engine. The overall conclusion of our study is that all three techniques, classification trees, artificial neural networks and fuzzy logic appeared to be reliable to predict macroinvertebrate communities in polluted streams.