Diluent composition for use of API 20E in characterizing marine and estuarine bacteria.

Nine chemically defined inoculation diluents, with compositions ranging from 0.85% NaCl to 35% marine salts, were used to evaluate the influence of diluent composition on the biochemical profiles of 30 marine and estuarine bacterial strains, including species of Vibrio, Aeromonas, Allomonas, and Photobacterium. Results demonstrated that a 20% marine salts diluent enabled the characterization of halophilic strains normally nonreactive by the API 20E system. Furthermore, the use of 20% marine salts showed that certain environmental isolates, identifiable as Vibrio parahaemolyticus by the recommended clinical inoculation procedure, were Vibrio vulnificus. An analysis of the profiles provided by the nine diluents indicates that the API 20E system, modified by the use of a diluent composed of 20% marine salts and incubated at 22 degrees C, can provide a reliable tool for the rapid characterization of marine and estuarine bacterial isolates.     

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

A. DALSGAARD, I. DALSGAARD, L. HØI AND J.L. LARSEN. 1996. Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus .     

Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region

Abstract An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus . The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.     

Comparison of different biochemical and molecular methods for the identification of Vibrio parahaemolyticus

AIMS: Multicentre evaluation of biochemical and molecular methods for the identification of Vibrio parahaemolyticus. METHODS AND RESULTS: For the biochemical identification methods, API 20E and API 20NE and Alsina's scheme were evaluated in intra- and interlaboratory tests in order to determine the accuracy and concordance of each method. Both in intra- and interlaboratory tests, the Alsina's scheme showed the highest sensitivity (86% of correct identifications in the interlaboratory test). False-positive results were obtained by all methods (specificity was 95% for API 20E, 73% for API 20NE and 84% for Alsina's scheme) and concordance varied from 65% of API 20NE to 84% of API 20E. For the molecular identifications, polymerase chain reaction (PCR) for the detection of toxR gene, tl gene and pR72H fragment were tested on 30 strains by two laboratories. The PCR for toxR showed the highest inclusivity (96%), exclusivity (100%) and concordance (97%). CONCLUSIONS: Among the biochemical identification methods tested, the Alsina's scheme gave more reliable results; however, in order to avoid false-positive results, all the biochemical identifications should be confirmed by means of molecular methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Availability of an efficient identification method of Vibrio parahaemolyticus to use in official control of fisheries products.     

Comparison of biochemical and molecular methods for the identification of bacterial isolates associated with failed loggerhead sea turtle eggs

Aims: Comparison of biochemical vs molecular methods for identification of microbial populations associated with failed loggerhead turtle eggs. Methods and Results: Two biochemical (API and Microgen) and one molecular methods (16s rRNA analysis) were compared in the areas of cost, identification, corroboration of data with other methods, ease of use, resources and software. The molecular method was costly and identified only 66% of the isolates tested compared with 74% for API. A 74% discrepancy in identifications occurred between API and 16s rRNA analysis. The two biochemical methods were comparable in cost, but Microgen was easier to use and yielded the lowest discrepancy among identifications (29%) when compared with both API 20 enteric (API 20E) and API 20 nonenteric (API 20NE) combined. A comparison of API 20E and API 20NE indicated an 83% discrepancy between the two methods. Conclusions: The Microgen identification system appears to be better suited than API or 16s rRNA analysis for identification of environmental isolates associated with failed loggerhead eggs. Significance and Impact of the Study: Most identification methods are not intended for use with environmental isolates. A comparison of identification systems would provide better options for identifying environmental bacteria for ecological studies.     

Molecular analysis of Vibrio parahaemolyticus isolated from human patients and shellfish during US Pacific north-west outbreaks

AIMS: The objective of this study was to investigate the occurrence and distribution of haemolysin genes, plasmid profile, serogroup analysis and cellular urease activity for Vibrio parahaemolyticus isolates from infected human patients and oysters from the Pacific north-western United States between 1988 and 1997. METHODS AND RESULTS: All of the clinical and environmental isolates tested in this study exhibited the presence of the thermolabile haemolysin gene, tl, confirming that all of the isolates were V. parahaemolyticus. Furthermore, the V. parahaemolyticus isolates that contained either the thermostable direct haemolysin gene, tdh, or the thermostable direct haemolysin-related gene, trh, or both, were also positive for urease. Isolates from infected human patients belong to serogroups O1 and O4, whereas, the isolates from oysters belong to serogroups O1, O4 and O5. These results suggest that the presence of a V. parahaemolyticus serogroup O1 and O4 could indicate the presence of a virulent strain of this pathogen. In this study, the presence of the haemolysin genes, serogroup profiles and urease production in V. parahaemolyticus isolated from human patients correlated with the oysters collected during the outbreaks. However, no significant correlation of the plasmid profiles was detected, based on their distribution and molecular weights, between V. parahaemolyticus isolated from infected human patients and from oysters collected during this outbreak. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: It is apparent from this study that the identification of the haemolysin genes by multiplex PCR amplification, in conjunction with serogroup analysis and urease production, can be used to monitor shellfish for the presence of potentially pathogenic strains of V. parahaemolyticus.     

Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus

The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed. An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.     

Oligonucleotide probes specific for the genus Salmonella and for Salm. typhimurium

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64·9% of environmental isolates. The E/NF system gave a positive identification to 88·0% of isolates and the 20E to 79·5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.     

Phenotypic and genetic analysis of Enterobacter spp. from a Brazilian oligotrophic freshwater lake

We characterized a population of Enterobacter spp. of the Enterobacter cloacae complex isolated from an oligotrophic lake; most isolates were identified as E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological, biochemical, physiological, and plasmid profiles analyses, including antimicrobial susceptibility testing, were performed on 22 environmental isolates. Misidentification occurred when using the API 20E identification system. Analysis of 16S rDNA sequences confirmed the close relatedness between species of the E. cloacae complex. The tDNA PCR allowed the differentiation and identification of the E. cloacae isolates. Evaluation of genetic diversity by 16S rDNA sequence, tDNA, internal transcribed spacers, and enterobacterial repetitive intergenic concensus profiles revealed nearly identical isolates, although they exhibited different physiological and antimicrobial resistance profiles. Among the Enterobacter isolates, 96% were resistant to at least one antimicrobial; multiple resistance was also found at a high frequency (86%). The antimicrobials against which resistance was found most frequently were beta-lactams, chloramphenicol, and streptomycin. Plasmids were found in 21 of the 22 Enterobacter isolates. This confirms the conception that antibiotic resistance can occur in oligotrophic freshwater lake bacteria, which has important implications for public health.     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Comparison of rapid NFT and API 20E with conventional methods for identification of gram-negative nonfermentative bacilli from pharmaceuticals and cosmetics.

The accuracy of the Rapid NFT and the API 20E identification systems was evaluated by comparing them with conventional biochemical methods for the identification of gram-negative, nonfermentative bacilli. The organisms were recovered from preserved, nonsterile pharmaceutical and cosmetic products. A total of 123 test isolates that are commonly encountered in these products were used. By using the criteria of accurate and reliable identification without employing additional tests, Rapid NFT was found to be more accurate after 48 h of incubation than API 20E for characterizing isolates to the species level. Therefore, close agreement between NFT and conventional methods for identification of industrial gram-negative isolates provides evidence that the Rapid NFT system is an improved and rapid method for identifying these organisms to the species level with minimal use of supplementary tests.     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.     

Ecology of Vibrio mimicus in aquatic environments

An environmental study was done to examine the prevalence of Vibrio mimicus in some aquatic environments of Dhaka, Bangladesh, and of Okayama, Japan. Water samples from Dhaka environments and water and plankton samples from Okayama environments were quantitatively as well as qualitatively analyzed throughout the seasons for V. mimicus. The organism was isolated from Bangladesh environments throughout the year, whereas it was not isolated in Okayama when the water temperature fell below 10 degrees C. Samples with as many as 9.0 x 10(2) CFU of V. mimicus per 100 ml of water in Dhaka and 1.5 x 10(4) CFU of V. mimicus per 100 ml of water in Okayama were detected during the study period. V. mimicus was not found in any environment with an average salinity of 10% or more. Brackish environments with an average salinity of 4% were observed to be the optimal natural condition for the pathogen. Using the API 20E system with the conventional test methods, we observed variations in biochemical properties within the V. mimicus species. This study reveals the inefficacy of the API 20E system to identify a significant percentage of V. mimicus. Therefore, in addition to the API 20E system, a salt tolerance test and a string test are recommended for identification of this species. Susceptibility testing of strains isolated from Okayama environments showed higher resistance to ampicillin and susceptibility to trimethoprim-sulfamethoxazole when compared with environmental isolates of V. mimicus from Bangladesh.     

Ecology of Vibrio mimicus in aquatic environments.

An environmental study was done to examine the prevalence of Vibrio mimicus in some aquatic environments of Dhaka, Bangladesh, and of Okayama, Japan. Water samples from Dhaka environments and water and plankton samples from Okayama environments were quantitatively as well as qualitatively analyzed throughout the seasons for V. mimicus. The organism was isolated from Bangladesh environments throughout the year, whereas it was not isolated in Okayama when the water temperature fell below 10 degrees C. Samples with as many as 9.0 x 10(2) CFU of V. mimicus per 100 ml of water in Dhaka and 1.5 x 10(4) CFU of V. mimicus per 100 ml of water in Okayama were detected during the study period. V. mimicus was not found in any environment with an average salinity of 10% or more. Brackish environments with an average salinity of 4% were observed to be the optimal natural condition for the pathogen. Using the API 20E system with the conventional test methods, we observed variations in biochemical properties within the V. mimicus species. This study reveals the inefficacy of the API 20E system to identify a significant percentage of V. mimicus. Therefore, in addition to the API 20E system, a salt tolerance test and a string test are recommended for identification of this species. Susceptibility testing of strains isolated from Okayama environments showed higher resistance to ampicillin and susceptibility to trimethoprim-sulfamethoxazole when compared with environmental isolates of V. mimicus from Bangladesh.     

Occurrence of pathogenic vibrios in coastal areas of France

AIMS: This study was carried out to investigate the occurrence of potentially pathogenic species of Vibrio in French marine and estuarine environments. METHODS AND RESULTS: Samples of coastal waters and mussels collected between July and September 1999 were analysed by culture, using selective media including thiosulphate-citrate-bile salts-sucrose and modified cellobiose-polymixin B-colistin agar. Presumptive Vibrio colonies were isolated and identified using selected biochemical tests. Specific primers based on flanking sequences of the cytolysin, vvhA gene, pR72H DNA fragment and 16S-23S rRNA intergenic spacer region (ISR) were used in a polymerase chain reaction (PCR) to confirm the identification of Vibrio vulnificus, V. parahaemolyticus and V. cholerae, respectively. In this study, V. alginolyticus (99 of 189) was the predominant species, followed by V. parahaemolyticus (41 of 189), V. vulnificus (20 of 189) and non-O1/non-O139 V. cholerae (three of 189). All 20 V. vulnificus isolates showed PCR amplification of the vvhA gene, 16 of which had been isolated from estuarine water. The PCR amplification of the pR72H DNA fragment in 41 V. parahaemolyticus isolates generated two unique amplicons of 387 and 320 bp. The latter, present in 24.4% of these isolates, had not previously been found in V. parahaemolyticus strains examined to date. Amplification of the trh gene in two of the isolates suggested these to be virulent strains. Three strains identified as V. cholerae by amplification of the 16S-23S rRNA ISR were confirmed to be non-cholera (non-O1/non-O139) strains. CONCLUSIONS: The results of this study demonstrated the presence of pathogenic Vibrio species in French coastal waters. Furthermore, the PCR approach proved useful for the rapid and reliable confirmation of species identification. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings indicate the potential sanitary risk associated with the presence of pathogenic Vibrio spp. in cultivated mussels and in the aquatic environment. The PCR can be used to detect pathogenic vibrios directly in environmental samples.     

Biochemical properties of Corynebacterium amycolatum strains

C. amycolatum is the most commonly isolated nonlipophilic species of Corynebacterium from clinical samples. However, the lack of good commercial identification tests in microbiology laboratories causes some difficulties in C. amycolatum diagnostics. We decided to examine biochemical and enzymatic properties of isolated strains and analyze the occurrence of particular biochemical profiles (biotypes). Perhaps it would let improve the identification schemes. 70 strains of C. amycolatum were analyzed. The estimation of biochemical properties consisted of the results of API Coryne and API ZYM tests (bioMérieux), the ability of excreting of protease, esterase, lipase and lecithinase. Analyzed strains had various biochemical and enzymatic properties. Almost all strains fermented glucose (98.6%) and maltose (95.7%) and produced pyrasinamidase (94.3%). All strains produced alkaline phosphatase and phosphohydrolase, and 95.7%--acid phosphatase. Biotypes of particular strains were determined on the biochemical reactions included in the API Coryne tests. In the group of 70 strains 21 profiles were distinguished among which 3100325 biotype (35.7%) was dominant. The lipolysis was defined on Tween 20, Tween 40, Tween 60, Tween 80 medium and with the API ZYM test usage. All strains produced esterase-lipase (esterase C-8), 95.7% of strains-esterase C-4, and 21.4% lipase C-14. Among analyzed strains 18.6% hydrolyzed Tween 20, 14.3% Tween 60, and 1.4% Tween 40. None of these strains demonstrated lipase and lecithinase activity. Difficulties in concerning C. amycolatum as pathogens justify further investigations.     

Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system

AIMS: To find the cause of misidentification of aeromonads when using the Vitek system. METHODS AND RESULTS: Two Aeromonas veronii biovar sobria isolates were misidentified as Vibrio alginolyticus by the Vitek system. Both strains' identification was confirmed by biochemical testing, API 20E/20NE kits and/or 16S RFLP analysis. Thirty-one known Aeromonas species were tested by the Vitek system using 0.45 and 0.85% saline in the suspension medium. It was not clear whether low salinity causes misidentification of Aeromonas species more frequently. CONCLUSIONS: The specified reaction time may be inappropriately short for some critical biochemical tests of some strains. An ingenious reading strategy regarding incubation time is necessary to improve identification of Aeromonas species by the Vitek system. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of misidentification of A. veronii biovar sobria as V. alginolyticus in the Vitek system.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)