Detection of Escherichia coli in water by culture-based amperometric and luminometric methods.

The application of amperometric biosensor- and chemiluminiscence based methods for rapid detection of viable E. coli in water has been investigated. An amplification of the amperometric signal by a factor of 4 was obtained when the cellobiose dehydrogenase (CDH) biosensor was used instead of a plain graphite electrode for detection of b-galactosidase (b-GAL) activity at 22.5 degrees C. A linear correlation was demonstrated for detection time (DT) vs. initial concentrations (logarithmic units) of E. coli IT1 and E. coli in environmental samples, respectively, by use of the CDH biosensor or a chemiluminometric technique. The study has shown that an E. coli concentration > or = 10(4) cfu/100 mL in environmental samples was determined by the CDH biosensor within one working day. However, further reduction of the DT can be obtained, e.g. by increasing the signal amplification factor using other biosensors.

Effect of u.v. light irradiation, starvation and heat on Escherichia coli beta-D-galactosidase activity and other potential viability parameters.

The effect of u.v. light irradiation and two other types of stress (heat and starvation) on cellular functions of Escherichia coli have been studied. The severe reduction of the culturable cell number (cfu) and the direct viable count (DVC) after exposure to moderate u.v. light doses (48 mWs cm-2), was not reflected by the dehydrogenase activity (5-cyano-2,3-ditolyl tetrazolium chloride (CTC)-positive cells), the membrane integrity (SYTOX Green-negative cells), the membrane potential (bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4[3]) (OXONOL)-negative cells), and the beta-D-galactosidase activity. All parameters were affected by high u.v. light doses. Cellular activities (CTC, SYTOX, OXONOL, beta-D-galactosidase activity) were intact in non-culturable cells with presumably severe damage to DNA, and the activities seemed not to be appropriate for detection of viable E. coli after u.v. light irradiation. Heating for 20-30 min at 63 degrees C was required to cause a severe loss of the beta-D-galactosidase activity and the numbers of CTC-positive, SYTOX Green-negative or OXONOL-negative cells. A large portion (> or = 38%) of pre-irradiated (190 mWs cm-2) cells maintained their ability to reduce CTC and exclude SYTOX Green and OXONOL after 51 d of starvation (dark, 7 degrees C) in phosphate-buffered saline.

Effect of chlorination on beta-D-galactosidase activity of sewage bacteria and Escherichia coli.

The effect of chlorine on beta-D-galactosidase activity of sewage bacteria and Escherichia coli was studied. beta-D-galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0.05 mg Cl2 l-1) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0.1 mg Cl2 l-1, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu-1 increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu-1 decreased when the chlorine dosage was increased from 0.1 to 0.5 mg Cl2 l-1, but were not significantly different (P > 0.05) for dosages of 0.2-0.7 mg Cl2 l-1. After chlorination, beta-D-galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.

Enzyme characteristics of beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria and their interference in rapid methods for detection of waterborne coliforms and Escherichia coli.

Bacteria which were beta-D-galactosidase and beta-D-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the beta-D-galactosidase-positive nontarget bacteria but none of the beta-D-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5 degrees C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria. The induced and noninduced beta-D-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced beta-D-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean beta-D-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

Effect of seawater on Escherichia coli beta-galactosidase activity.

An investigation of beta-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. beta-Galactosidase activity was induced by isopropyl-beta-D-thiogalactoside (IPTG). Enzyme activity (U cell-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell-1 = -8.5 whereas uninduced E. coli yielded log U cell-1 = -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in beta-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that beta-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, beta-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.

Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques.

Enzyme assays for 4-methylumbelliferyl-beta-D-galactopyranosidase and 4-methylumbelliferyl-beta-D-glucuronidase activities were used for rapid detection (25 min) of fecal water pollution and to determine the impact of sewage discharge in coastal waters. Two coastal areas were investigated: (i) an estuary characterized by a high degree of contamination downstream of a discharge from a sewage treatment plant and a low degree of water renewal and (ii) a fjord with a low degree of pollution and a high degree of water renewal. Statistical analysis showed that a global correlation curve could be used to estimate concentrations of culturable fecal coliform bacteria in the two coastal areas, although environmental factors important for cell physiology (e.g., salinity) varied at different sampling locations. The sensitivity limit for detection of 4-methylumbelliferyl-beta-D-glucuronidase activity corresponded to bacterial concentrations on the order of 10 to 100 CFU/100 ml. The 4-methylumbelliferyl-beta-D-galactopyranosidase assay was less sensitive because of a higher rate of substrate autohydrolysis. The detection limit corresponded to bacterial concentrations on the order of 100 to 1,000 fecal coliforms per 100 ml.

Production of 4-methylumbelliferyl heptanoate hydrolase by Escherichia coli exposed to seawater.

The production of an enzyme, 4-methylumbelliferyl heptanoate hydrolase, in Escherichia coli exposed to enriched and nonenriched seawater was studied. In all media, except for seawater with no or very small amounts of organic material and seawater enriched with peptone, 4-methylumbelliferyl heptanoate hydrolase activity increased by 2 to 3 orders of magnitude within 2 days. Increased enzyme activity was assumed to be related to cells not undergoing lysis but adapting to conditions of nutrient limitation.

Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-beta-D-galactoside.

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.

Evaluation of a fluorescent antibody technique for the rapid enumeration of Bacteroides fragilis group of organisms in water.

The Bacteroides fragilis group has been evaluated as a prospective rapid indicator of faecal contamination of water. Fluorescent antibody (FA) stained B. fragilis group bacteria were enumerated microscopically and compared with faecal coliform or Escherichia coli counts as indicators of faecal contamination. Environmental samples included surface waters (raw drinking water and known contaminated water). Laboratory disinfection experiments with ozone, chlorine and u.v. radiation were also performed. Bacteroides FA counts specifically detected recent human faecal contamination in field samples in 2-3 h. Samples with a high content of particulates or debris limited the sensitivity to about 10 FA counts/ml. Viable counts showed that the sensitivity to all three disinfection agents was essentially the same for Bacteroides and E. coli. Fluorescent antibody counts of Bacteroides, conversely, were not altered by any of the agents. Therefore, the Bacteroides FA method is not recommended for routine monitoring but may be useful for cases where extensive human faecal contamination is suspected (e.g. pipeline rupture or pollution of recreational water) and where rapid remedial action must be taken to protect the public health.

Survival and detection of Bacteroides spp., prospective indicator bacteria.

Preliminary experiments were performed to assess the use of intestinal Bacteroides spp. as indicators of fecal contamination of water. Viable counts of Bacteroides fragilis, an anaerobic bacterium, declined more rapidly than those of Escherichia coli and Streptococcus faecalis. However, a fluorescent antiserum prepared against B. fragilis successfully detected high proportions (18 to greater than 50%) of B. fragilis cells suspended for 8 days in aerobic water in dialysis bags at the ambient temperature. These percentages were higher than the percent viable recoveries of the two indicator bacteria used for comparison. Thus, the fluorescent antiserum test for B. fragilis might serve as a useful indicator of fecal contamination of water. An advantage of this approach over coliform analysis is the rapidity at which the test can be performed.