MICROBIOLOGICAL DETECTION OF E. COLI WITH UNPARALLELED SENSITIVITY. RUG A novel beta-glucuronidase substrate

Transcription

1 MICROBIOLOGICAL DETECTION OF E. COLI WITH UNPARALLELED SENSITIVITY RUG A novel beta-glucuronidase substrate

2 E. coli detection Escherichia coli (E. coli) is a Gram negative bacterium that inhabits the intestines of warm-blooded animals and is always found in feces. The presence of E. coli is the most important indicator of fecal contamination of drinking water, surface water, food and beverages. In addition, some strains of E. coli are pathogenic.

3 E. coli detection E. coli SUBSTRATE (colorless or faint color) β-d-glucuronidase COLOR, FLUORESCENCE Living cells of E. coli can be reliably detected with media that contain a chromogenic or fluorogenic substrate for beta-glucuronidase (also termed β-d-glucuronidase), an enzyme that occurs almost exclusively in E. coli. 03

4 Conventional detection of beta-glucuronidase activity using MUG MUG (4-Methylumbelliferyl beta-d-glucuronide) is commonly used as a substrate for detecting beta-glucuronidase activity in food, drinking water, clinical samples and for rapid bacterial identification in blood cultures. MUG is recommended in international guidelines (ISO, DIN) as a fluorogenic substrate for the detection of E. coli. 4-Methylumbelliferyl-beta- D-glucuronic acid dihydrate Biosynth Cat.No. M-5700 Assay: Fluorescence of bacteria culture Different bacteria were incubated in lauryl sulfate broth added with 120 mg/l MUG (M-5700), 24 h 37 C. 1. Escherichia coli ATCC Salmonella enteritides RKI 05/ Pseudomonas aeruginosa DSMZ Sterile control Picture was taken under UV light. 04

5 RUG novel substrate Resorufin-beta-D-glucuronic acid methyl ester Resorufin is one of the most intensely fluorescent materials known until today. Hence Resorufin-beta-D-glucuronide represents the perfect agent to detect E. coli bacteria at very low concentrations. Until recently, the application of this reagent was limited by low availability and high costs. At Biosynth s microbiology lab we discovered that the methyl ester analog Resorufin-beta-D-glucuronide methyl ester (RUG ) performs exactly the same as the free acid reagent Resorufin-beta-D-glucuronide. 1. SUBSTRATE ACTIVATION 2. ENZYME-SUBSTRATE REACTION Ester hydrolysis β-d-glucuronidase mediated by: - a medium component, or - an esterase, or - other ester hydrolysing effects Resorufin-β-D-glucuronic acid methyl ester Resorufin-β-D-glucuronic acid Resorufin The reaction scheme: 1. The methyl group is removed by media components or esterase activity, thus generating the desired free acid reagent Resorufin-beta-D-glucuronide in vivo. 2. E.coli specific beta-galactosidase activity subsequently catalyzes the hydrolysis of Resorufin-beta-D-glucuronic acid to Resofurin. Resofurin is a water soluble fluorescent dye with pink coloration that is easily detected visually on plates, membrane filters or in solution. 05

6 RUG simplifies the process of E. coli detection THE PRODUCT: Resorufin-beta-D-glucuronic acid methyl ester (RUG ) Biosynth Patent applied: EP Biosynth Cat. No. R-2155_P00 COLORATION AFTER 22 H: RUG generates a strong pink coloration - No UV light needed In contrast to MUG, RUG does not require fluorescence detection, as the released dye Resofurin is of intense pink color. Fluorescence detection is optional. Fluorescence of Resofurin is excited at 565 nm in the range of red light and does not require UV illumination. S.enteritides E. coli sterile control Assay: Escherichia coli ATTC and Salmonella enteritides RKI 05/07992 were inoculated at low density (20 CFU / ml) in AT E. coli detection broth containing 12 mg/l R-2155_P00 (RUG ). Tubes were incubated at 37 C and 150 rpm. 06

7 Rapid color development - RUG outperforms MUG KINETICS IN MICROTITER PLATES START WITH 10 CFU E.COLI PER WELL Fluorescence (RFU) RUG MUG The speed of light The full Resofurin signal develops in incubation times as short as 10 hrs, even at very low bacteria concentration in a given sample Time (h) Assay: E. coli ATCC was inoculated at 10 CFU/well in nutrient broth containing either 0.1 mm RUG or 0.1 mm MUG. The microtiter plate was incubated at 37 C and red fluorescence (left, RUG ) or blue fluorescence (right, MUG) was recorded. 07

8 RUG is 10 times more sensitive as compared to MUG FLUORESCENCE AFTER 24H. E. COLI GROWN IN LAURYN SULFATE BROTH 100 Relative fluorescence (%) RUG MUG RUG is a highly efficient beta-glucoronidase substrate for the detection of E.coli: A concentration of only 12 mg/l of RUG in the growth media generates a 100% of the fluorescence signal Concentration of RUG and MUG [mg/l] Assay: E. coli was incubated for 24 h in lauryl sulfate broth containing concentrations of 0.9 mg/l to 120 mg/l of either RUG or MUG. Red fluorescence (left, RUG ) or blue fluorescence (right, MUG) was recorded. 08

9 RUG is economic Using RUG instead of MUG is an economic decision even though there are higher costs of purchase for the top-notch substrate RUG. Here are the reasons: Low concentration of substrate: MUG needs a 10-fold higher concentration to develop the full signal. As a result the costs for one assay using the conventional substrate MUG is equal to one assay using the innovative substrate RUG. Considerably shorter incubation times: E. coli is detected after only 10 hours of incubation using RUG as fluorogenic/ chromogenic substrate No UV light required: Using RUG E.coli enzyme activity will be detected by a pink coloration of the media 09

10 RUG is economic (continued) Blue auto-fluorescence of Pseudomonas spp. does not interfere with the red fluorescence of Resofurin E. coli E. coli + RUG TM Pseudomonas spec. + Cefsulodin pink color red fluorescence + MUG blue fluorescence blue fluorescence With RUG no additional antibiotic is required: Conventional MUG creates a blue fluorescence signal in E. coli assays that can not be distinguished readily from natural background caused by auto-fluorescence of Pseudomonas spp. Therefore, the high-grade antibiotic Cefsulodin (Biosynth Cat.No.s C-2490, C-2492) is typically added in MUG assays to specifically inhibit the growth of Pseudomonas spp. Using RUG in the application allows you to skip this step as RUG gives a pink color and a red fluorescence signal. 10

11 Fluorophore Resorufin is stable in a wide ph range The maximum fluorescence of 4-Methylumbelliferone is reached at a ph value of 9. But at physiological ph values the fluorescence is only 10% - 20%. In contrast, the fluorescence of Resofurin is highest at ph value 8; at ph value 6.5 the fluorescence signal still gives 50% of the maximum fluorescence. Relative fluorescence (%) Resofurin 4-Methyl-umbelliferone Resofurin red fluorescence in a test tube ph Assay: Robust fluorescent signal: Effect of ph values on the fluorescent signals of Resofurin and 4-Methylumbelliferone. Fluorescence was excited at respective optimum wavelengths 565 nm and 366 nm. Using RUG instead of MUG gives the researcher more flexibility in terms of the media composition and ph range of the media. 11

12 RUG is the new MUG THE ADVANTAGES OF RESORUFIN-BETA-D-GLUCURONIC ACID METHYL ESTER SUBSTRATE AT A GLANCE: Chromogenic and fluorogenic beta-glucuronidase substrate Easy to detect pink colored end product Fluorescent detection is optional and does not require UV illumination 10-fold more sensitive as compared to MUG Fluorescence emission at ph values Cost efficient: low substrate concentration, no additional antibiotics Please visit for more fluorogenic and chromogenic enzyme substrates BIOSYNTH AG BIOSYNTH AG, Rietlistr. 4, CH-9422 Staad, Switzerland