Water and biofilm in drinking water distribution systems in the Netherlands.

To keep the high quality of drinking water in the future for non-chlorinated drinking water systems, knowledge about the variables that most strongly affect this quality is necessary in order to know where to focus on and possibly even change aspects of drinking water production and distribution. Therefore, the aim of this study was to investigate which variables (source of drinking water, growth potential and pipe material type) have the biggest influence on bacterial community composition and biomass concentration of drinking water and biofilm in distribution systems. Ten different distribution systems were sampled for water and biofilm, obtained from four different pipe materials, throughout the Netherlands. The distribution systems are supplied either with drinking water produced from groundwater or surface water, and differ in drinking water quality parameters such as the growth potential. We found a significant relationship for growth potential and ATP concentration in water, but for the ATP in the biofilm none of the parameters showed a significant effect. Furthermore, the source of the drinking water and the pipe material did not significantly affect the ATP concentration in water and biofilm. The bacterial composition of in both water and biofilm was significantly different between distribution systems delivering water with low and high growth potential and between drinking water produced from groundwater or surface water. In contrast, the different pipe materials did not significantly affect composition of biofilm-associated communities. We conclude from these results that the growth potential of the treated water best explains the variation in biomass and bacterial composition in water and biofilm of non-chlorinated drinking water distribution systems followed by the drinking water source, whereas pipe materials seem to be of lesser importance.

Influence of pipe materials on the microbial community in unchlorinated drinking water and biofilm.

Biodegradable compounds can cause undesired microbial growth in drinking water systems and these compounds can originate from the water or pipe materials used in drinking water systems. The aim of our study was to determine the influence of different pipe materials on the microbial populations in water and biofilm under semi-stagnant conditions. The microbial communities in biofilm and water, which were in contact with seven different materials, were characterized by determining ATP concentrations, microbial composition gene copy numbers of some specific microbial groups. The ATP concentration in water and biofilm varied between the different materials with glass (negative control) < copper < PVCC < PE-Xc < PE-Xb < PE-100 < PVC-P. Gene copy numbers of Legionella spp., Mycobacterium spp., Pseudomonas spp., Aeromonas spp., fungi and Vermamoeba vermiformis were also higher for PVC-P and PE than for glass, copper and PVCC. The bacterial community composition in water and biofilm varied between materials as well. PERMANOVA and CAP analysis demonstrated that copper and PVC-P are different when compared to the other materials. Furthermore, bacterial community composition and ATP concentrations in water and biofilm were similar after eight and 16 weeks incubation, but differed from results obtained after one week. Finally, the ATP, the specific microbial groups and the bacterial community composition also differed between water and biofilm on each material. We conclude from our study that pipe material is an important factor that influences the biomass concentration, abundance of specific microorganisms and the bacterial community composition in distribution systems with unchlorinated drinking water.

Limited presence of Waddlia chondrophila in drinking water systems in the Netherlands.

Waddlia chondrophila is an emerging pathogen belonging to the order of Chlamydiales. This obligate intracellular bacterium was initially isolated from an aborted bovine fetus and is associated with adverse pregnancy outcomes in women. The ability of W. chondrophila to reside and replicate within a range of free-living amoebae implies a possible widespread environmental presence. Potential hosts of W. chondrophila are present in Dutch drinking water. This study therefore investigated the presence of W. chondrophila DNA in drinking water by analysing 59 samples from ten drinking water systems throughout the Netherlands. Samples were taken at three distances from the treatment plant, during both summer and winter. Twelve of the samples were positive, originating from two of the treatment plants, of which three samples were quantifiable.

Legionella growth potential of drinking water produced by a reverse osmosis pilot plant.

Treatment processes, such as membrane filtration with reverse osmosis (RO), are used to produce drinking water with a high degree of biostability. To our knowledge, the influence of RO water on biofilm formation and growth of L. pneumophila has not yet been investigated. Therefore, this study aimed (i) to determine the Legionella growth potential of (remineralised) RO-water produced by a pilot plant and to compare this to conventional treated groundwater, and (ii) to determine if different pipe materials, in contact with remineralised RO-water, can cause growth of L. pneumophila. The Legionella growth potential of water was determined with the boiler biofilm monitor (BBM) that mimics the flow of water in a premise plumbing system. The Legionella growth potential of materials in contact with remineralised RO-water was determined by using the biomass production potential (BPP)-test. ATP concentrations in the biofilm on the glass rings from the BBM fed with (remineralised) RO water fluctuated around 100 pg ATP cm-2. In contrast, BBMs fed with conventionally treated water resulted in ten-fold higher ATP concentrations in the biofilm. Moreover, conventionally treated water had a Legionella growth potential that was 1000-fold higher than that of (remineralised) RO-water. Furthermore, glass, copper and PVC-C had the lowest biofilm concentrations and Legionella growth potential in the BPP-test, followed by PE-Xb, PE-Xc and PE-100. The highest biofilm concentration and Legionella growth potential were with PVC-P. Hence, our study demonstrated that remineralised RO-water did not enhance growth of L. pneumophila in the BBM that mimics the premises plumbing system. However, when PE or PVC-P materials are used growth of L. pneumophila can still occur in the premises plumbing system despite the high quality of the supplied remineralised RO-water.

Slowly biodegradable organic compounds impact the biostability of non-chlorinated drinking water produced from surface water.

It is possible to distribute drinking water without a disinfectant residual when the treated water is biologically stable. The objective of this study was to determine the impact of easily and slowly biodegradable compounds on the biostability of the drinking water at three full-scale production plants which use the same surface water, and on the regrowth conditions in the related distribution systems. Easily biodegradable compounds in the drinking water were determined with AOC-P17/Nox during 2012-2015. Slowly biodegradable organic compounds measured as particulate and/or high-molecular organic carbon (PHMOC), were monitored at the inlet and after the different treatment stages of the three treatments during the same period. The results show that PHMOC (300-470 µg C L-1) was approximately 10% of the TOC in the surface water and was removed to 50-100 µg C L-1. The PHMOC in the water consisted of 40-60% of carbohydrates and 10% of proteins. A significant and strong positive correlation was observed for PHMOC concentrations and two recently introduced bioassay methods for slowly biodegradable compounds (AOC-A3 and biomass production potential, BPC14). Moreover, these three parameters in the biological active carbon effluent (BACF) of the three plants showed a positive correlation with regrowth in the drinking water distribution system, which was assessed with Aeromonas, heterotrophic plate counts, coliforms and large invertebrates. In contrast, the AOC-P17/Nox concentrations did not correlate with these regrowth parameters. We therefore conclude that slowly biodegradable compounds in the treated water from these treatment plants seem to have a greater impact on regrowth in the distribution system than easily biodegradable compounds.

Secretory component mediates Candida albicans binding to epithelial cells.

OBJECTIVES: Candida albicans attaches to oral surfaces via a number of mechanisms including adherence mediated by salivary components adsorbed to the C. albicans cell surface. Our goal was to identify the salivary molecules involved. MATERIALS AND METHODS: Biotinylated salivary polypeptides that were bound by C. albicans were detected in extracts from washed, saliva-treated yeast cells by polyacrylamide gel electrophoresis and electroblot or immunoblot transfer analysis and purified by electroelution. Purified material was tested for the ability to promote the adherence of radiolabelled C. albicans yeast cells to cultured epithelial monolayers. RESULTS: Three of the polypeptides bound by C. albicans cells were identified as components of secretory IgA, including secretory component. Using non-denaturing polyacrylamide gel electrophoresis, we demonstrated that secretory component could be detected in its free form in saliva, and was bound by yeast cells. Secretory component which was purified by electroelution from non-denaturing PAGE-separated saliva, without detectable complete IgA, promoted adherence of yeast cells to cultured epithelial monolayers in a dose-dependent fashion. CONCLUSION: These results indicate that despite the inhibitory effect on adherence of IgA specific to C. albicans, IgA components, in particular secretory component, also promote binding to cultured epithelial monolayers.

Adherence of Candida albicans to silicone is promoted by the human salivary protein SPLUNC2/PSP/BPIFA2.

Interactions between Candida albicans, saliva and saliva-coated oral surfaces are initial events in the colonization of the oral cavity by this commensal yeast, which can cause oral diseases such as candidiasis and denture stomatitis. Candida albicans also colonizes silicone voice prostheses, and the microbial biofilm formed can impair valve function, necessitating frequent prosthesis replacement. We have previously shown that saliva promoted binding of C. albicans cells to silicone in vitro, and that the selective binding of specific salivary proteins to voice prosthesis silicone mediated attachment of C. albicans cells. The C. albicans cells adhered to a polypeptide (or polypeptides) of ~36 kDa eluted from saliva-treated silicone. We show here that a protein of similar size was identified in replicate blots of the eluate from saliva-treated silicone when the blots were probed with antibodies to human SPLUNC2, a salivary protein with reported microbial agglutination properties. In addition, SPLUNC2 was depleted from saliva that had been incubated with silicone coupons. To determine whether SPLUNC2 is a yeast-binding protein, SPLUNC2 cDNA was expressed in Escherichia coli. Purified recombinant His-tagged protein (SPLUNC2r) bound to silicone as demonstrated by immunoblot analysis of an eluate from SPLUNC2r-treated silicone coupons and (35) S-radiolabelled C. albicans cells adhered in a dose-dependent manner to SPLUNC2r-coated silicone. We conclude that SPLUNC2 binds to silicone and acts as a receptor for C. albicans adherence to, and subsequent colonization of, voice prosthesis silicone.

Assessment of nitrification in groundwater filters for drinking water production by qPCR and activity measurement.

In groundwater treatment for drinking water production, the causes of nitrification problems and the effectiveness of process optimization in rapid sand filters are often not clear. To assess both issues, the performance of a full-scale groundwater filter with nitrification problems and another filter with complete nitrification and pretreatment by subsurface aeration was monitored over nine months. Quantitative real-time polymerase chain reaction (qPCR) targeting the amoA gene of bacteria and archaea and activity measurements of ammonia oxidation were used to regularly evaluate water and filter sand samples. Results demonstrated that subsurface aeration stimulated the growth of ammonia-oxidizing prokaryotes (AOP) in the aquifer. Cell balances, using qPCR counts of AOP for each filter, showed that the inoculated AOP numbers from the aquifer were marginal compared with AOP numbers detected in the filter. Excessive washout of AOP was not observed and did not cause the nitrification problems. Ammonia-oxidizing archaea grew in both filters, but only in low numbers compared to bacteria. The cell-specific nitrification rate in the sand and backwash water samples was high for the subsurface aerated filter, but systematically much lower for the filter with nitrification problems. From this, we conclude that incomplete nitrification was caused by nutrient limitation.

Utilization of oligo- and polysaccharides at microgram-per-litre levels in freshwater by Flavobacterium johnsoniae.

AIMS: To obtain a bacterial strain that can be used to quantify biodegradable polysaccharides at concentrations of a few micrograms per litre in freshwater. METHODS AND RESULTS: Flavobacterium johnsoniae strain A3 was isolated from tap water supplemented with laminarin, pectin or amylopectin at 100 microg C l(-1) and river Rhine water. The organism utilized 14 of 23 oligo- and polysaccharides, and 1 of 9 monosaccharides, but none of the sugar acids, sugar alcohols, carboxylic acids or aromatic acids tested at 10 microg C l(-1). Amino acids promoted growth of strain A3, but not in coculture with assimilable organic carbon (AOC) test strain Pseudomonas fluorescens P17, which utilized these compounds more rapidly than strain A3. Compounds released by strain P17 and AOC test strain Spirillum sp. NOX grown on acetate promoted the growth of strain A3 at N(max) values of > or = 2 x 10(5) CFU ml(-1) of strain P17 and > or = 5 x 10(5) CFU ml(-1) of strain NOX. Significant growth of strain A3 was observed in surface water and in tap water in the presence of strain P17 (N(max) P17 < 2 x 10(5) CFU ml(-1)). CONCLUSIONS: Strain A3 utilizes oligo- and polysaccharides at microgram-per-litre levels. In surface water and in tap water, the organism was able to utilize compounds that were not utilized by strain P17. These compounds may include oligo- and/or polysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytoplanktonic and bacterial polysaccharides can constitute an important biodegradable fraction of natural organic matter in water and may promote growth of heterotrophic bacteria during water treatment and drinking water distribution. Strain A3 can be used to quantify a group of compounds that includes oligo- and polysaccharides at microgram-per-litre levels in freshwater.

Modelling the length of microbiological protection zones around phreatic sandy aquifers in The Netherlands.

The aim of the current study was to calculate the size of protection zones around (sub)oxic and anoxic sandy aquifers without confining layers using a virus infection and transport model. The maximum allowable virus infection risk was 10(-4)/person/year at the 95% confidence level. Model results demonstrated that phreatic (sub)oxic sandy aquifers in The Netherlands required protection areas with a residence time of 43-117 d to ensure that the maximum virus infection risk would not be exceeded. This was 0.7-2 x the current guideline of 60d. In contrast, phreatic anoxic sandy aquifers without confining layers needed protection zones of 555-898d to stay below the maximum virus infection risk, 9.5-15 x the current guideline. A sensitivity analysis of the model demonstrated that the calculated protection zone was most sensitive for virus inactivation rate and collision efficiency. Values of both parameters were predicted from values obtained from previously published field and laboratory studies. At present, as it is unknown if these values can also be used at other locations, model results should be interpreted with care.

Effects of twisting of the graft in anterior cruciate ligament reconstruction.

In an in vitro study on six knees from cadavers, the effect of bone-patellar tendon-bone graft twist on anterior knee laxity was measured at different knee flexion angles. A motion and loading rig was used to prescribe the flexion angle, to restrain axial rotation, and to apply 100 N anterior force to the tibia. Roentgen stereophotogrammetric analysis was used to measure the relative anteroposterior position of the tibia and femur. The tibial bone block was cemented in a cylinder that allowed rotation in the bone tunnel. The anterior cruciate ligament was transected and reconstructed with neutral, 90 degrees, and 150 degrees internal twists and 90 degrees and 150 degrees external twists. External and internal graft twists in the reconstruction resulted in significant reductions of anterior laxity, however, at the cost of a more posterior position of the unloaded tibia relative to the femur (anteroposterior-error). The results are explained by the anterior relocation of the graft insertion by twisting the tibial bone block. The inclination angle of the graft in the anteroposterior direction flattens, which could improve the anterior laxity. A consequent side effect is the increase of posterior shift of the tibia relative to the femur.

Effect of administration of Lactobacillus crispatus, Clostridium lactatifermentans and dietary lactose on the development of the normal microflora and volatile fatty acids in the caeca of broiler chicks.

1. Lactobacillus crispatus and Clostridium lactatifermentans, both isolated from the caeca of chickens, grown together in an in vitro model system are able to ferment lactose to acetate and propionate. In this study, the capabilities of these organisms were studied in vivo. 2. The effect on concentrations of volatile fatty acids and lactate, together with the development of some bacterial groups in the caeca of chicks, was studied after oral inoculation with L. crispatus and C. lactatifermentans together with dietary lactose. For this purpose, chicks were divided into 4 groups: (i) control group, (ii) dietary lactose, (iii) L. crispatus and C. lactatifermentans, and (iv) dietary lactose together with L. crispitaus and C. lactatifermentans. 3. In general, concentrations of (undissociated) volatile fatty acids in the caeca were not significantly different in broilers receiving both bacteria and dietary lactose compared with control broilers. Concentrations of lactate in the caeca of 14-d-old broilers treated with any of the three treatments were significantly higher than in the caeca of control broilers. 4. This indicates that L. crispatus or other lactate-producing organisms were responsible for the elevated concentrations of lactic acid. Clostridium lactatifermentans has probably not colonised the caeca sufficiently to ferment this lactate further to acetate and propionate. 5. Numbers of Enterobacteriaceae and enterococci in the caeca of broilers receiving both bacteria and dietary lactose were not different from control broilers. 6. We conclude from these results that under the conditions applied in this study a mixture of L. crispatus and C. lactatifermentans with dietary lactose was able to increase lactate concentrations but was unable to increase concentrates of acetate and propionate in the caeca of broiler chicks.

Spatial and temporal variation of the intestinal bacterial community in commercially raised broiler chickens during growth.

The objective of this study was to determine whether host, compartment, or environmental specific factors play an important role in the establishment of the intestinal microflora in broiler chickens during growth. This objective was addressed using a 16S rDNA approach. PCR-amplicons from the V6 to V8 regions of the 16S rDNA of intestinal samples were separated by denaturing gradient gel electrophoresis (DGGE). The number of bands in all intestinal compartments increased when broilers grew older, indicating that the dominant bacterial community becomes more complex when chickens age. Each chicken had a unique banding pattern for all locations in the intestinal tract, irrespective of the age of chickens. This suggests that host-related factors affect the establishment of the dominant bacterial community. Banding patterns of intestinal compartments within one chicken were different from each other for broilers older than 4 days, except for both ceca which were highly similar. In 4-day-old broilers, banding patterns from crop, duodenum, and ileum were very similar. We conclude that (unknown) host specific factors play an important role in the development of the intestinal bacterial community in each broiler chicken. Furthermore, compartment-specific factors play an important role in the bacterial development of each intestinal compartment within one chicken.

Inhibition of a glucose-limited sequencing fed-batch culture of Salmonella enterica serovar enteritidis by volatile fatty acids representative of the ceca of broiler chickens.

The effects of concentrations of volatile fatty acids on an anaerobic, glucose-limited, and pH-controlled growing culture of Salmonella enterica serovar Enteritidis were studied. Suddenly increasing volatile fatty acids to the concentrations representative of the ceca of 15-day-old broiler chickens caused washout of serovar Enteritidis. In contrast, a sudden increase to the volatile fatty acid concentrations representative of the ceca of younger broiler chickens caused a reduction in the biomass but not washout. Gradually increasing volatile fatty acids caused a gradual decrease in the biomass of serovar Enteritidis. We conclude that the concentrations of volatile fatty acids present in the ceca of broilers with a mature microflora can cause washout of serovar Enteritidis in an in vitro system mimicking cecal ecophysiology.

Role of volatile fatty acids in development of the cecal microflora in broiler chickens during growth.

It is known that volatile fatty acids can inhibit growth of species of the family Enterobacteriaceae in vitro. However, whether these volatile fatty acids affect bacterial populations in the ceca of chickens is unknown. Therefore, a study was conducted to investigate if changes in volatile fatty acids in ceca of broiler chickens during growth affect bacterial populations. Results showed that members of the Enterobacteriaceae and enterococci are present in large numbers in 3-day-old broilers and start to decrease when broilers grow older. Lactobacilli are present in large numbers as well in 3-day-old broilers, but they remain stable during the growth of broilers. Acetate, butyrate, and propionate increase from undetectable levels in 1-day-old broilers to high concentrations in 15-day-old broilers, after which they stabilize. Significant negative correlations could be calculated between numbers of Enterobacteriaceae and concentrations of undissociated acetate, propionate, and butyrate. Furthermore, pure cultures of Enterobacteriaceae isolated from the ceca were grown in the presence of volatile fatty acids. Growth rates and maximal optical density decreased when these strains grew in the presence of increasing volatile fatty acid concentrations. It is concluded that volatile fatty acids are responsible for the reduction in numbers of Enterobacteriaceae in the ceca of broiler chickens during growth.