Presence and persistence of wastewater pathogen Escherichia coli O157:H7 in hydroponic reactors of treatment wetland species.

Treatment wetlands (TWs) efficiently remove many pollutants including a several log order reduction of pathogens from influent to effluent; however, there is evidence to suggest that pathogen cells are sequestered in a subsurface wetland and may remain viable months after inoculation. Escherichia coli is a common pathogen in domestic and agricultural wastewater and the O157:H7 strain causes most environmental outbreaks in the United States. To assess attachment of E. coli to the TW rhizosphere, direct measurements of E. coli levels were taken. Experiments were performed in chemostats containing either Teflon nylon as an abiotic control or roots of Carex utriculata or Schoenoplectus acutus. Flow of simulated wastewater through the chemostat was set to maintain a 2 hour residence time. The influent was inoculated with E. coli O157:H7 containing DsRed fluorescent protein. Root samples were excised and analyzed via epifluorescent microscopy. E. coli O157:H7 was detected on the root surface at 2 hours after inoculation, and were visible as single cells. Microcolonies began forming at 24 hours post-inoculation and were detected for up to 1 week post-inoculation. Image analysis determined that the number of microcolonies with >100 cells increased 1 week post-inoculation, confirming that E. coli O157:H7 is capable of growth within biofilms surrounding wetland plant roots.

Floating treatment wetlands for domestic wastewater treatment.

Floating islands are a form of treatment wetland characterized by a mat of synthetic matrix at the water surface into which macrophytes can be planted and through which water passes. We evaluated two matrix materials for treating domestic wastewater, recycled plastic and recycled carpet fibers, for chemical oxygen demand (COD) and nitrogen removal. These materials were compared to pea gravel or open water (control). Experiments were conducted in laboratory scale columns fed with synthetic wastewater containing COD, organic and inorganic nitrogen, and mineral salts. Columns were unplanted, naturally inoculated, and operated in batch mode with continuous recirculation and aeration. COD was efficiently removed in all systems examined (>90% removal). Ammonia was efficiently removed by nitrification. Removal of total dissolved N was ∼50% by day 28, by which time most remaining nitrogen was present as NO(3)-N. Complete removal of NO(3)-N by denitrification was accomplished by dosing columns with molasses. Microbial communities of interest were visualized with denaturing gradient gel electrophoresis (DGGE) by targeting specific functional genes. Shifts in the denitrifying community were observed post-molasses addition, when nitrate levels decreased. The conditioning time for reliable nitrification was determined to be approximately three months. These results suggest that floating treatment wetlands are a viable alternative for domestic wastewater treatment.

Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes.

AIMS: To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs). METHODS AND RESULTS: Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify dehII genes from selected indicator strains. The developed primer sets were effective in directly amplifying dehII genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the dehII gene was used. CONCLUSIONS: This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify dehII genes in drinking water systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.

Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms.

AIMS: The hypothesis that surrogate planktonic pathogens (Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested. METHODS AND RESULTS: Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0.15 and 1.5 N m(-2). The amount of surrogate pathogens introduced ranged from approximately 10(5) CFU ml(-1) to 10(10 )spheres ml(-1). The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0.4-3.0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated. CONCLUSIONS: Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.

Retention of a model pathogen in a porous media biofilm.

The inadvertent or the deliberate introduction of pathogens into drinking water can lead to public health consequences. Distribution system sampling strategies are needed to provide information on the identity, source and fate of the introduced pathogens. Porous media biofilm reactors conditioned with undefined drinking water biofilms were tested for their ability to immobilize Escherichia coli 0157:H7. Biofilms were established by applying continuous flow of biologically activated carbon treated water with natural microflora and supplemented nutrient solution (0.5 mg l(-1) C) for 2 or 3 weeks. Control reactors were clean and were not colonized with biofilm. All reactors were injected with slug doses of approximately 1 x 10(9) cfu E. coli O157:H7. On the basis of the plate count enumeration of the introduced pathogen, reactors pre-colonized for 2 or 3 weeks retained significantly more cells (0.75 and 9.37% of the introduced spike dose, respectively) compared with uncolonized control reactors (0.22%). Compared with cultivation, microscopic direct counts and quantitative PCR suggested significantly higher and lower numbers of pathogens, respectively. Plate counts were thus considered as the method of choice for pathogen enumeration in this study. In addition to providing general insights into interactions between pathogens and drinking water biofilms, the study concluded that engineered biofilm systems may be considered as a device to capture pathogens from the bulk flow for monitoring purposes.

Denaturing gradient gel electrophoresis can rapidly display the bacterial diversity contained in 16S rDNA clone libraries.

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35-50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (approximately 1500 bp) than was actually analyzed in DGGE (approximately 350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.

Interaction of Klebsiella oxytoca and Burkholderia cepacia in dual-species batch cultures and biofilms as a function of growth rate and substrate concentration.

Dual-species microbial interactions have been extensively reported for batch and continuous culture environments. However, little research has been performed on dual-species interaction in a biofilm. This research examined the effects of growth rate and substrate concentration on dual-species population densities in batch and biofilm reactors. In addition, the feasibility of using batch reactor kinetics to describe dual-species biofilm interactions was explored. The scope of the research was directed toward creating a dual-species biofilm for the biodegradation of trichloroethylene, but the findings are a significant contribution to the study of dual-species interactions in general. The two bacterial species used were Burkholderia cepacia PR1-pTOM(31c), an aerobic organism capable of constitutively mineralizing trichloroethylene (TCE), and Klebsiella oxytoca, a highly mucoid, facultative anaerobic organism. The substrate concentrations used were different dilutions of a nutrient-rich medium resulting in dissolved organic carbon (DOC) concentrations on the order of 30, 70, and 700 mg/L. Presented herein are single- and dual-species population densities and growth rates for these two organisms grown in batch and continuous-flow biofilm reactors. In batch reactors, planktonic growth rates predicted dual-species planktonic species dominance, with the faster-growing organism (K. oxytoca) outcompeting the slower-growing organism (B. cepacia). In a dual-species biofilm, however, dual-species planktonic growth rates did not predict which organism would have the higher dual-species biofilm population density. The relative fraction of each organism in a dual-species biofilm did correlate with substrate concentration, with B. cepacia having a greater proportional density in the dual-species culture with K. oxytoca at low (30 and 70 mg/L DOC) substrate concentrations and K. oxytoca having a greater dual-species population density at a high (700 mg/L DOC) substrate concentration. Results from this research demonstrate the effectiveness of using substrate concentration to control population density in this dual-species biofilm.

Assessing technician effects when extracting quantities from microscope images.

Consider an experiment where the response is based on an image; e.g., an image captured to a computer file by a digital camera mounted on a microscope. Suppose relevant quantitative measures are extracted from the images so that results can be analyzed by conventional statistical methods. The steps involved in extracting the measures may require that the technicians, who are processing the images, perform some subjective manipulations. In this case, it is important to determine the bias and variability, if any, attributable to the technicians' decisions. This paper describes the experimental design and statistical analyses that are useful for those determinations. The design and analysis are illustrated by application to two biofilm research projects that involved quantitative image analysis. In one investigation, the technician was required to choose a threshold level, then the image analysis program automatically extracted relevant measures from the resulting black and white image. In the other investigation, the technician was required to choose fiducial points in each of two images collected on different microscopes; then the image analysis program registered the images by stretching, rotating, and overlaying them, so that their quantitative features could be correlated. These investigations elucidated the effects of the technicians' decisions, thereby helping us to assess properly the statistical uncertainties in the conclusions for the primary experiments.

Movement, replication, and emigration rates of individual bacteria in a biofilm.

Single-cell behavior within a biofilm was observed over a period of several hours. The observations were converted into quantitative stochastic rules governing the behavior of individual cells within a biofilm. Such a quantitative summary provides not only a concise description of the results but also information helpful when constructing computer models of dynamic biofilm systems. The time to division, emigration, and rate of motility of individual green fluorescent protein labeled Pseudomonas aeruginosa PAO1 cells in a 3-10 microm thick biofilm containing predominantly non-GFP labeled cells were calculated based on images of individual cells collected at 15-min time intervals. The biofilms were grown in flow cells and the images captured with a confocal laser microscope. Cells destined to emigrate are more active than those that remain; the geometric means for velocities in the biofilm are 1.0 microm/h for remaining cells and 1.5 microm/h for emigrating cells. The median time to emigration was 2.0 h. During the experimental observation period, the estimated probability for emigration is 0.44, illustrating that a substantial number of bacteria leave the field of view. Cells emigrate at a median time one-third that of the median time to replication. Specifically, the median time for cells to divide was 6.9 h, and it was estimated that 10% of the cells had a time to division greater than 10 h.

Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth.

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P. putida and pure C(12)-HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.

Apparent surface associated lag time in growth of primary biofilm cells.

The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions.

Effect of growth conditions and substratum composition on the persistence of coliforms in mixed-population biofilms.

Laboratory reactors operated under oligotrophic conditions were used to evaluate the importance of initial growth rate and substratum composition on the long-term persistence of coliforms in mixed-population biofilms. The inoculum growth rate had a dramatic effect on the ability of coliforms to remain on surfaces. The most slowly grown coliforms (mu = 0.05/h) survived at the highest cell concentration. Antibody staining revealed that Klebsiella pneumoniae existed primarily as discrete microcolonies on the surface. Both coliforms and heterotrophic plate count bacteria were supported in larger numbers on a reactive substratum, mild steel, than on polycarbonate.

Effects of Motility and Adsorption Rate Coefficient on Transport of Bacteria through Saturated Porous Media.

Three strains of Pseudomonas fluorescens with different motility rates and adsorption rate coefficients were injected into porous-medium reactors packed with l-mm-diameter glass spheres. Cell breakthrough, time to peak concentration, tailing, and cell recovery were measured at three interstitial pore velocities (higher than, lower than, and much lower than the maximal bacterial motility rate). All experiments were done with distilled water to reduce the effects of growth and chemotaxis. Contrary to expectations, motility did not result in either early breakthrough or early time to peak concentration at flow velocities below the motility rate. Bacterial size exclusion effects were shown to affect breakthrough curve shape at the very low flow velocity, but no such effect was seen at the higher flow velocity. The tendency of bacteria to adsorb to porous-medium surfaces, as measured by adsorption rate coefficients, profoundly influenced transport characteristics. Cell recoveries were shown to be correlated with the ratio of advective to adsorptive transport in the reactors. Adsorption rate coefficients were found to be better predictors of microbial transport phenomena than individual characteristics, such as size, motility, or porous-medium hydrodynamics.

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.

Sensitivity of genetically engineered organisms to selective media.

Eighteen strains of Escherichia coli used in genetic studies were tested for their ability to grow on several selective media. Highest recoveries were obtained with m-T7 agar. The SOS system, particularly the recA gene, may play some role in the sensitivity of E. coli to selective agents. These results may be important in the selection of media used to detect genetically engineered organisms released into the environment.

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.

Growth and persistence of pathogens on granular activated carbon filters.

Three enteric pathogens Yersinia enterocolitica O:8, Salmonella typhimurium, and enterotoxigenic Escherichia coli, were examined for their ability to colonize granular activated carbon (GAC) in pure cultures and in the presence of autochthonous river water organisms. All three organisms readily colonized sterile GAC and maintained populations of ca. 10(5) to 10(7) CFU g-1 for 14 days when suspended in sterile river water. Exposure of pathogen biofilms on GAC to unsterile river water resulted in a gradual decline in pathogens on the carbon (0.08 to 0.14 log day-1). When pathogens were introduced to sterile GAC in the presence of heterotrophic plate count organisms, they attached at levels similar to those in the pure cultures and then decreased (0.10 to 0.22 log day-1). When added with heterotrophic plate count bacteria to GAC supporting a mature biofilm of native river water bacteria, they attached at a lower level (1.0 X 10(4) to 4.6 X 10(4) CFU g-1) and decreased at a more rapid rate (0.11 to 0.70 log day-1).

Disinfection of bacteria attached to granular activated carbon.

Heterotrophic plate count bacteria, coliform organisms, and pathogenic microorganisms attached to granular activated carbon particles were examined for their susceptibility to chlorine disinfection. When these bacteria were grown on carbon particles and then disinfected with 2.0 mg of chlorine per liter (1.4 to 1.6 mg of free chlorine residual per liter after 1 h) for 1 h, no significant decrease in viable counts was observed. Washed cells attached to the surface of granular activated carbon particles showed similar resistance to chlorine, but a progressive increase in sublethal injury was found. Observations made by scanning electron microscope indicated that granular activated carbon was colonized by bacteria which grow in cracks and crevices and are coated by an extracellular slime layer. These data suggest a possible mechanism by which treatment and disinfection barriers can be penetrated and pathogenic bacteria may enter drinking water supplies.

Evaluation of m-T7 agar as a fecal coliform medium.

m-T7 agar, designed to improve recoveries of injured total coliforms, was evaluated for its effectiveness as a fecal coliform medium. The time and temperature of preincubation were found to be crucial to the optimal recovery of fetal coliforms. Isolation rates for fecal coliforms on m-T7 agar from sewage effluents were the highest when plates were preincubated at 37 degrees C for 8 h before transfer to 44.5 degrees C for 12 h. The medium was found to produce consistently higher fecal coliform counts than all the other methods tested. Recoveries were 3.1 times greater than the standard m-FC method and 1.7 times greater than the two-layer enrichment, temperature acclimation procedure. Verification rates for fecal coliforms isolated on m-T7 agar averaged 89.0%, whereas verification rates for m-FC agar averaged only 82.8%. Both media isolated similar fecal coliform populations. The advantages of a single medium, highly effective for the isolation of both total and fecal coliforms, are discussed.

Enumeration of indicator bacteria exposed to chlorine.

Stress resulting from a variety of chemical and physical environments has been recognized in indicator bacteria. A review by Busta (1976) summarizes the extensive work that has been carried out to describe indicator microorganisms sublethally impaired due to a variety of causes associated with foods. Workers in the area of water microbiology are also gaining an appreciation of the importance of these stressed cells in the assessment of water quality using bacterial indicators. Chemical agents, including chlorine, that are employed in water disinfection processes are important causes of bacterial stress injury. As a result, a significant portion of the total population of indicator bacteria in water might not be enumerated (using the selective procedures that are currently employed) and inaccurate water quality determinations could result. Alternative water disinfection agents that are being suggested, such as ozone, chlorine dioxide, and ultraviolet irradiation, will also probably lead to the same result. In addition, heat from thermal pollution and interactions with other microorganisms or chemicals (including disinfectants and metals) also exert stress that could further debilitate indicator bacteria in various waters and effluents. A need for improved enumeration procedures has accompanied the recognition of injured indicator bacteria in chlorinated waters and wastewaters. This movement has also stimulated interest in the underlying mechanism of cellular damage that is responsible for the submaximal recovery of coliforms from disinfected waters. Various groups have reported that a number of biochemical, genetic, and physiological processes are impaired by chlorine exposure under differing conditions. Evidence from our laboratory and elsewhere implicates functions associated with the cell envelope, i.e., the uptake of extracellular organic substrates, as the primary cellular target of chlorine under conditions that are similar to those in the field. Additional data from our group indicate that sublethal damage from chlorine can be reversed under suitable nonselective conditions. Recent efforts have led to the development of new methods to enumerate injured fecal streptococcus, total and fecal coliform bacteria from chlorinated waters and wastewater. These procedures each yield data that are comparable with that obtained using the more cumbersome MPN method. As a result, the best characteristics of both methods may now be found in three relatively simple MF procedures. Some of these advances have been described in a new section (#921) of the fifteenth edition of "Standard Methods for the Examination of Water and Wastewater" entitled "Stressed Organisms" (APHA, 1981). However, it is anticipated that new and better water quality assessment methodologies will emerge from the growing literature concerning the physiological and biochemical behavior of indicator microorganisms in water and wastewater.(ABSTRACT TRUNCATED AT 400 WORDS)