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Inhibitory effect of esculin on oxidative DNA damage and carcinogenesis induced by N-nitrosobis(2-oxopropyl)amine in hamster pancreas.

The effects of esculin, a natural coumarin compound, on the formation of 8-oxo-2'-deoxyguanosine (8-oxodG) and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were given a diet containing esculin for 7 days, and killed 4~h after BOP treatment, and the contents of 8-oxodG were measured in the nuclear DNA of the pancreas. Esculin suppressed significantly the increase in the 8-oxodG content of hamster pancreas induced by BOP. Furthermore, the effect of esculin on the rapid production model experiment for pancreatic carcinogenesis using BOP was investigated. Esculin was given ad libitum as a 0.05% aqueous solution during either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly lower than in the control group, while the incidence in animals given esculin during the promotion phase showed no significant change. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.

8-Hydroxy-2'-Deoxyguanosine↗

Esculin hydrolysis by Vibrio vulnificus.

A clinical isolate of Vibrio vulnificus was found to hydrolyze esculin when tested on bile-esculin-azide agar during the initial characterization of the strain. Reports in the literature of esculin hydrolysis by V. vulnificus are conflicting. We tested herein 52 strains of V. vulnificus from clinical and environmental sources for the ability to hydrolyze esculin. Seventy-eight percent of the strains hydrolyzed esculin on bile-esculin-azide agar, whereas all strains of V. vulnificus tested were positive for esculin hydrolysis in a noninhibitory medium, whereas some strains failed to hydrolyze esculin on media containing inhibitory compounds.

Animals↗

Esculin hydrolysis by Enterobacteriaceae.

Literature reports disagree concerning esculin hydrolysis in the family Enterobacteriaceae. A total of 2,490 strains of the family were investigated for esculin hydrolysis by two methods, the esculin spot test and the PathoTec incubation strip, which measures constitutive enzyme, and five growth-supporting methods, which determine both constitutive and inducible enzymes. The five growth-supporting media studied were: Vaughn-Levine, the standard esculin hydrolysis medium (P. R. Edwards and W. H. Ewing, Identification of Enterobacteriaceae, 3rd ed., 1972); Vaughn-Levine without iron; Vaughn-Levine without Andrade's indicator; and bile-esculin medium. Growth media were incubated at 35 degrees C and checked every 24 h for 120 h. On growth media, 0.3% of Escherichia coli were positive in 24 h, 34% in 48 h, and 61% in 120 h. No strains were positive on the "nongrowth" tests. It appeared that the esculin hydrolysis enzyme(s) of E. coli was inducible rather than constitutive. All esculin hydrolyzers, which yielded positive tests on "constitutive tests" and 24-h tests, were limited to the genera Klebsiella, Enterobacter, and Serratia and species of Proteus vulgaris, Proteus rettgeri, and Citrobacter diversus. When used with standardized inoculum size and incubation time, the esculin hydrolysis test is very useful for differentiation within the family Enterobacteriaceae.

Citrobacter↗

Suppression of 8-oxo-2'-deoxyguanosine formation and carcinogenesis induced by N-nitrosobis (2-oxopropyl)amine in hamsters by esculetin and esculin.

Effects of esculetin (6,7-dihydroxycoumarin) and its glycoside, esculin, on 8-oxo-2'-deoxyguanosine (8-oxodG) formation and carcinogenesis induced by a chemical carcinogen, N-nitrosobis(2-oxopropyl)amine (BOP), were examined in the pancreas of female Syrian golden hamsters. Animals were administered esculetin by gastric intubation into the stomach 30 min before BOP administration or ingestion of a diet containing esculin for 7 days before BOP administration, and killed 1 or 4h after BOP treatment, and the contents of thiobarbituric acid-reacting substrates (TBARS) and 8-oxodG in the pancreas were determined. Both compounds suppressed significantly the BOP-induced increases in 8-oxodG and TBARS contents in hamster pancreas. We further investigated the effect of esculin on pancreatic carcinogenesis by the rapid production model induced by augmentation pressure with a choline-deficient diet, ethionine, methionine and BOP. Esculin was given ad libitum as a 0.05% aqueous solution in either the initiation or promotion phases. The incidence of invasive tumors in animals given esculin during the initiation phase was significantly smaller than in the control group, while esculin given during the promotion phase showed no apparent effects. These results suggest that the intake of esculin has an inhibitory effect on BOP-induced oxidative DNA damage and carcinogenesis in hamster pancreas.

8-Hydroxy-2'-Deoxyguanosine↗

Esculin hydrolysis by Gram positive bacteria. A rapid test and it's comparison with other methods.

A number of bacteria hydrolyze esculin enzymatically to esculetin. This characteristic is used by taxonomists and clinical microbiologists in the differentiation and identification of bacteria, especially to distinguish Lance-field group D streptococci from non-group D organisms and Listeria monocytogenes from morphologically similar Erysipelothrix rhusipoathiae and diphtheroids. Conventional methods used for esculin hydrolysis require 4--48 h for completion. We developed and evaluated a medium which gives positive results more rapidly. The 2,330 isolates used in this study consisted of 1,680 esculin positive and 650 esculin negative organisms. The sensitivity and specificity of this method were compared with the PathoTec esculin hydrolysis strip and the procedure of Vaughn and Levine (VL). Of the 1,680 esculin positive organisms, 97% gave positive reactions within 30 minutes with the rapid test whereas PathoTec required 3--4 h incubation for the same number of organisms to yield a positive reaction.

Bacteria↗

Esculin hydrolysis reaction by Escherichia coli.

The literature contains variable reports concerning the hydrolysis of esculin by members of the family Enterobacteriaceae and particularly Escherichia coli. We examined 113 strains of fresh clinical isolates of E. coli and assessed the ability of colonies in a population to hydrolyze esculin with and without preincubation in inducible substrates at 24, 48, and 72 h. The number of strains capable of fermenting salicin, a sugar with a beta-glucoside linkage like esculin, was studied under the same conditions. A strip test that measured the presence of the constitutive glucosidase was also performed with and without preincubation in inducible substrates. No E. coli strain was able to produce constitutive enzyme; preincubation in esculin and salicin resulted in an induction of the beta-glucosidase. The number of colonies able to hydrolyze esculin increased with time. Only those strains preincubated in esculin or salicin were able to produce a positive constitutive strip test. Because the beta-glucosidase of E. coli is inducible, one should employe, when using growth media, a light inoculum obtained by touching the top of a colony with a bacteriological wire and read the reaction between 18 and 24 h, or perform a rapid strip or spot test.

Benzyl Alcohols↗

[The description of an esculin-positive biovar of Pseudomonas aeruginosa].

In the study of 280 P. aeruginosa strains isolated in different hospitals of St. Petersburg for the first time 48 strains capable of hydrolyzing esculin have been detected. The hydrolysis of esculin is determined in plates with the use of the microvolume techniques the results were evaluated after 3-hour incubation at 37 degrees C. The data confirming the existence of the exculin-positive biovar of P. aeruginosa have been obtained; these data show the wide spread of esculin-positive strains in hospitals of different specialization (17.1 +/- 5.1% of P. aeruginosa strains), the characteristic combination of the sign of esculin hydrolysis with such signs as the absence of the smell of trimethylamine and the phenomenon of "iridescent lysis" of the colonies, the stability of the sign of esculin hydrolysis in strains, repeatedly isolated from patients, after the storage of the cultures and their treatment with plasmid-eliminating preparation. The name "esculinolytica" has been proposed for this biovar. The typing strain of biovar esculinolytica has been deposited in the culture collection of the Russian Research Institute of Agricultural Microbiology as P. aeruginosa ARRIAM 64-A. This biovar been found to be most widely spread in urological hospitals, where esculin-positive strains are isolated 3 times more frequently (32.2 +/- 5.1% of P. aeruginosa strains) than in surgical hospitals (10.7 +/- 2.2%).

Culture Media↗

[Antibiotics sensitivity and characteristics of the esculin-positive Pseudomonas aeruginosa biovar].

Strains of Pseudomonas aeruginosa hydrolyzing esculin were isolated for the first time. They amount to 17.1 +/- 2.0% (60 from 325) of the investigated P. aeruginosa strains isolated from the clinical material in St. Petersburg. Esculin hydrolysis was measured by micromethod in plates, results were analysed after 3-hours incubation at 37 degrees C. Esculin-positive strains possesed biovar properties: they are widely spread, demonstrated other characteristic features (absence of triethylamine odour, specific colonies lysis), are stable on ability to hydrolyse esculin while culture storage and after repeated culturing. Typical strain of esculinolytica biovar was deposited into the culture collection of the National Research Institute of Agricultural Microbiology as P. aeruginosa ARRIAM 64-A. Susceptibility testing of the esculin-positive strains by disk-diffusion method revealed that most strains were inhibited by imipenem (86.6%), amikacin (75.0%), ceftazidime (65.0%), meropenem (60.0%), aztreonam (51.6%). The percent of strains susceptible to other antibiotics was lower: azlocillin--33.3%, netilmycin--33.3%, piperacillin--26.6%, ceftriaxon--18.3%. Only small number of strains were inhibited by ciprofloxacin (8.3%), gentamycin (3.4%), cefoperazone (1.7%) and carbenicillin (1.7%). The results may be used for empiric therapy before the isolated strain susceptibility is tested but only according to positive esculin-hydrolysis express-test evaluated in 3-hours period.

Anti-Bacterial Agents↗

The use of bile - esculin agar for the taxonomic classification of the family Enterobacteriaceae.

Bile-esculin medium has been used for many years for the presumptive identification of group D Streptococcus. The test is based on the ability of a bacterium to grow in the presence of 40% bile and produce esculinase. 2935 strains of Enterobacteriaceae were inoculated onto bile-esculin agar slants and incubated at 35 C. Esculin hydrolysis was determined after 24 and 48 hours. At 24 hours of incubation esculin hydrolysis was limited to the genera Klebsiella, Enterobacter, Serratia, and the species P. vulgaris, P. rettgeri, and C. diversus. Not all strains of these species were positive, however. All other members of the family were negative. At 48 hours of incubation 37% of E. coli gave a positive reaction; all other Enterobacteriaceae which were negative at 24 hours remained negative. Esculin hydrolysis is a valuable test for the taxonomic classification of the family Enterobacteriaceae.

Bacteriological Techniques↗

Esculine, ranitidine and carbenoxolone: different modes of action on gastric mucosa.

1. This study was designed to determine the antiulcerogenicity of esculine in various types of experimentally induced gastric ulcers in which the appearance of lesions is due to an ischemic process: cold-restraint stress and pylorus-ligated induced ulcers. 2. In the first experimental model, esculine (50 mg/kg) produced a significant diminution not only in the number of haemorrhagic stomachs (21.5% by 37.5% of the controls) but also in the ulcer index, U.I. (1.00 +/- 0.63, P less than 0.05). 3. When the mucosal damage was induced as a consequence of the pylorus-ligated gastric secretion, pretreatment of esculine (25 and 50 mg/kg) prevented the formation of gastric lesions (12.4 +/- 2.8, P less than 0.05 and 12.2 +/- 1.20, P less than 0.005), although it was less effective than ranitidine (2.8 +/- 1.8, P less than 0.025). However a significant reduction on the acidity with a dose of 25 mg/kg was observed (31.69 +/- 6.42, P less than 0.025). For the rest of the studied parameters: pepsin, histamine and Na and K electrolytes no differences with regard to the control groups were produced. 4. The effects of esculine on mucosal lesions produced by intragastric instillation of 1 ml of absolute ethanol, were also studied. In this model esculine did not show any protective effect and high U.I. values were obtained.

Animals↗

Use of bile-esculin agar for rapid differentiation of Enterobacteriaceae.

Bile-esculin agar has been used for several years for the presumptive identification of group D streptococci. All members of the Enterobacteriaceae family will also grow on this medium, but only certain ones can hydrolyze esculin to 6,7-dihydroxycoumarin, which reacts with iron to produce a characteristic blackening of the medium. One thousand and six cultures from clinical specimens representing 20 genera were isolated and identified. Heavy inocula from fresh pure culture isolates on heart infusion agar were placed on bile-esculin agar slants and incubated at 35 C. The slants were examined at 4 h and again at 18 h for esculin hydrolysis. Shigella, Salmonella, Arizona, Proteus mirabilis, Proteus morganii, Providencia alcalifaciens, and Providencia stuartii all produced negative results. Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens, and Serratia rubidaea produced a positive reaction in 4 h. The other remaining eight genera exhibited varying results. The use of this medium in conjunction with triple sugar iron-lysine iron agar has been of great value in differentiating the Klebsiella-Enterobacter-Serratia group from other Enterobacteriaceae.

Agar↗

Esculin-based medium for isolation and identification of Cryptococcus neoformans.

A simple medium was developed, using esculin as the substrate, for the isolation and identification of Cryptococcus neoformans. C. neoformans produced a brown-black pigment on the medium; all other yeasts produced no pigment or were light yellow. Esculin is beta-glucose-6,7-dihydroxycoumarin. C. neoformans produced pigment because the 6,7-dihydroxycoumarin component of the esculin molecule was converted to a melanin-like pigment. We think the reaction was similar to the conversion of diphenols, aminophenols, and diaminobenzenes to melanin. Laboratory studies with isolates of C. neoformans, C. albidus, C. luteolus, and C. terreus and representatives of the genera Candida, Torulopsis, Geotrichum, and Rhodotorula, plus environmental field studies, demonstrated that over 95% of C. neoformans isolates were correctly identified, whereas all other fungi were excluded. Esculin agar was a sensitive, specific medium for the isolation and identification of C. neoformans. It was inexpensive and had a long storage life.

Cryptococcus↗

Rapid spot test for the determination of esculin hydrolysis.

Esculin hydrolysis is a useful test in the differentiation of both gram-positive and gram-negative bacteria covering a wide spectrum of aerobes, facultative anaerobes, and anaerobes. Commonly utilized methods require a minimum of 18 h of incubation in broth or agar medium and utilize the production of a brown-black compound, due to the combination of ferric ions with the hydrolysis product esculetin, as indicator. A procedure is presented that requires 15 to 30 min for completion and utilizes fluorescence loss as the indicator of hydrolysis. Esculin fluoresces at 366 nm, whereas the hydrolysis product esculetin does not. Over 1,400 strains of gram-positive and gram-negative bacteria were tested. There was 98.4% of correlation between the spot test and esculin broth and 97% correlation with the bile-esculin agar.

Aerobiosis↗

Petite colony formation by Listeria monocytogenes and Listeria species grown on esculin-containing agar.

Several strains of Listeria species formed petite-sized colonies from parent stock cultures when grown on agar media containing 0.2-1% (w/v) esculin. This was observed in Listeria monocytogenes (7/22 strains), L. innocua (1/3), L. grayi (1/1), L. seeligeri (1/3), and L. welshimeri (1/1), but not in L. ivanovii (0/1) and L. murrayi (0/1). This phenomenon was only observed on agar media that contained esculin. All petite isolates had biotyping profiles identical to their larger, normal-sized counterpart isolates. Normal and petite-sized isolates from two L. monocytogenes strains, Scott A and V7, were pathogenic to immunosuppressed white mice. On media containing 0.5% (w/v) esculin + ferric iron, Listeria cultures produced colony diameters intermediate in size between those of normal and petite cultures. When pregrown in glucose broth, all petite isolates demonstrated visible beta-glucosidase (esculinase) activity within 5 min, while the normal-sized isolates showed beta-glucosidase activity only after at least 20-70 min. This evidence suggests that cells forming petite colonies are beta-glucosidase constitutive variants within the parent population, while cells that form normal-sized colonies are inducible for beta-glucosidase (esculinase) activity. A possible role for the esculin hydrolysis product, esculetin, in causing petite colony formation is discussed.

Animals↗

Non-aqueous capillary electrophoresis for separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its medicinal preparations.

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five samples.

Acetates↗

Rapid tests for esculin hydrolysis by anaerobic bacteria.

Esculin hydrolysis is one of the biochemical tests used in the identification of anaerobic microorganisms. The conventional method by use of growing microbial cells requires 24-48 hours of incubation. On the other hand, growth independent methods like the buffered esculin test, the spot test, and the PathoTec strip test utilize the presence of constitutive enzymes and, therefore, yield results in 1-4 hours. A total of 817 anaerobic organisms were used in this study to determine the sensitivity and specificity of the three rapid methods. All three rapid methods gave excellent correlation with the standard conventional method. Over 99% of the organisms gave comparable results with the spot test and the buffered esculin test within one hour; the PathoTec strip test required up to 4 hours. The former two were not only more rapid but also more economical than the PathoTec strip test.

Anaerobiosis↗

Effects of esculin and esculetin on the survival of Escherichia coli O157 in human faecal slurries, continuous-flow simulations of the rumen and colon and in calves.

The human pathogen Escherichia coli O157:H7 is thought to be spread by direct or indirect contact with infected animal or human faeces. The present study investigated the effects of the plant coumarin esculin and its aglycone esculetin on the survival of a strain of E. coli O157 under gut conditions. The addition of these compounds to human faecal slurries and in vitro continuous-flow fermenter models simulating conditions in the human colon and rumen caused marked decreases in the survival of an introduced strain of E. coli O157. When four calves were experimentally infected with E. coli O157 and fed esculin, the pathogen was detected in five of twenty-eight (18 %) of faecal samples examined post-inoculation, compared with thirteen of thirty-five (37 %) of faecal samples examined from five control calves not fed esculin. Coumarin compounds that occur naturally in dietary plants or when supplemented in the diet probably inhibit the survival of E. coli O157 in the gut.

Animals↗

Comparison of spot esculin hydrolysis with the PathoTec strip test for rapid differentiation of anaerobic bacteria.

The ability of several anaerobic bacteria to hydrolyze esculin to esculetin is used by clinical microbiologists and taxonomists in the differentiation and identification of both gram-positive and gram-negative microorganisms. Conventional methods used for determining esculin hydrolysis by anaerobic bacteria require 24 to 48 h for completion. In this paper we evaluate two procedures which yield rapid results. A total of 738 anaerobic bacteria were used in this study. A total of 99% of the esculin-hydrolyzing anaerobic bacteria gave positive results with the spot test in 1 h, whereas the other test method, the PathoTec strip test (General Diagnostics, Morris Plains, N.J.), required 4 h for 96% of the strains tested to yield positive reactions. Both tests showed a 100% specificity when compared with the standard broth test and are easy to perform, accurate, and economical. The spot test is superior to the PathoTec strip test in yielding results more rapidly.

Anaerobiosis↗