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.     

Identification of strains isolated as total and fecal coliforms and comparison of both groups as indicators of fecal pollution in tropical climates

This study was undertaken to better characterize the groups of total coliforms (TC) and fecal coliforms (FC) and to evaluate both groups as indicators of fecal contamination of drinking well water in a tropical climate (The Ivory Coast, West Africa). Isolated colonies obtained as TC or FC on membrane filters were identified using the API-20E system. From the well water samples, 58 golden-green colonies with a metallic sheen isolated on Endo medium (TC) were identified as Escherichia coli (55%), Enterobacter (26%), Klebsiella (14%), Proteus (3%), and Citrobacter (2%). Among 132 colonies isolated on Endo medium as non-TC (not showing the characteristic golden metallic sheen), 10% were identified as E. coli. The 196 blue colonies isolated on M-FC medium at 44.5 degrees C (FC) were identified as E. coli (66%), Klebsiella (12%), Enterobacter (10%), Citrobacter (5%), Salmonella (3%), Serratia (3%), Proteus (2%), and Yersinia (0.5%). Among 24 nonblue colonies on M-FC medium, none were identified as E. coli. Of the colonies isolated from human feces, E. coli represents 92% of the TC and 89% of the FC. Although these results are limited, they tend to confirm the greater specificity of the fecal coliform technique over that of total coliform for the detection of fecal contamination of untreated well water. From the results presented here and the observations of other workers, it is suggested that the use of FC instead of TC should be considered as the method of choice for determining drinking water pollution of untreated groundwater supplies.     

Cadmium tolerance and antibiotic resistance in Escherichia coli isolated from waste stabilization ponds

The incidence pattern of cadmium tolerance and antibiotics resistance by Escherichia coli was examined periodically from the samples of water, sludge and intestine of fish raised in waste stabilization ponds in a sewage treatment plant. Samples of water and sludge were collected from all the selected ponds and were monitored for total counts of fecal coliform (FC), total coliform (TC) and the population of Escherichia coli, which was also obtained from the intestine of fishes. Total counts of both FC and TC as well as counts of E. coli were markedly reduced from the facultative pond to the last maturation pond. Tolerance limit to cadmium by E. coli tended to decline as the distance of the sewage effluent from the source increased; the effective lethal concentration of cadmium ranged from 0.1 mM in split chamber to 0.05 mM in first maturation pond. E. coli isolated from water, sludge and fish gut were sensitive to seven out of ten antibiotics tested. It appears that holistic functions mediated through the mutualistic growth of micro algae and heterotrophic bacteria in the waste stabilization ponds were responsible for the promotion of water quality and significant reduction of coliform along the sewage effluent gradient.     

用污染指示菌评价城市水体污染的研究

选择湖南省常德市江北城区水体为代表开展本项研究。结果表明,水体中污染指示菌,总大肠菌群（ＴＣ）和粪大肠菌群（ＦＣ）密度同ＢＯＤ＿５负荷以及综合污染指数Ｐ呈高度正相关。所以,污染指示菌指标,特别是粪大肠菌群指标足综合评价城市污水,尤其是生活污水污染的一个必不可少的重要参数。对该城区水体细菌污染的评价结果是,生活污水排放愈集中的地区,细菌污染愈严重。其中护城河水的ＴＣ密度超出我国地面水Ⅲ级标准４个数量级;ＦＣ密度超过ＷＨＯ娱乐用水标准３个数量级。护城河是细菌污染最严重水体。包括滨湖公园内湖在内的接纳生活污水多或较多的水体,细菌污好也相当严重。只是远离城区的柳叮湖受污染程度较轻。     

Faecal indicator bacteria at fish farms

The observed concentrations of bacteria at two large fish farms were not high, but due to the great volume of the discharge the total amount of bacteria was large. Total coliform (TC) bacteria identified belonged mainly to the genera Enterobacter, Citrobacter and Aeromonas. The majority of faecal coliform (FC) strains were Escherichia coli. E. coli was absent, or occurred at very low concentrations, in the influent water, but was present in the effluent water, in the sediment, and at one fish farm also in fish faeces. FC bacteria were not observed in the fish feed. The concentrations of faecal streptococci (FS) in the influent water were low, but strains isolated were identified as group D streptococci. The concentrations of FS were low in the feed and sediment samples but were elevated in the fish faeces and also in the effluent.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Rapid seven-hour fecal coliform test.

A rapid 7-h fecal coliform (FC) test for the detection of FC in water has been developed. This membrane filter test utilizes a lightly buffered lactose-based medium (m-7-h FC medium) combined with a sensitive pH indicator system. FC colonies appeared yellow against a light purple background after incubation at 41.5 degrees C for 7 to 7.25 h. Comparison of FC test results showed that the mean verified FC count ratio (7-h FC count/24-h FC count) for surface water samples was 1.08. The mean FC count ratio (7-h FC count/24-h FC count) for unchlorinater wastewater ranged from 1.95 to 5.05. Verification of yellow FC colonies from m-7-h FC medium averaged 97%. Data from field tests on Lake Michigan bathing beach water samples showed that unverified 7-h FC counts averaged 96% of the 24-h FC counts. The 7-h FC test was found to be suitable for the examination of surface waters and unchlorinated sewage and could serve as an emergency test for detection of sewage or fecal contamination of potable water.     

Effect of sunlight on enumeration of indicator bacteria under field conditions.

The effect of sunlight on the enumeration of fecal coliform (FC) and fecal streptococcal (FS) bacteria when water samples are collected in containers and brought back to the laboratory for analysis or when the water samples are filtered through membranes on site was determined. FC and FS in raw sewage stored in clear glass or translucent polyethylene containers were resistant to the effects of sunlight. However, under the same conditions of storage and exposure to sunlight, 90% of FC and FS in sewage diluted 1:100 in seawater were inactivated within 13 to 32 min. When sewage was similarly diluted in stream water and exposed to sunlight, 90% of FC were inactivated after 28 to 38 min, whereas 90% of FS were not inactivated even after a 2-h exposure to sunlight. Other experiments showed that 90 to 99% of FC and FS retained on membranes were inactivated when these membranes were exposed to sunlight for 10 to 15 min. FS were inherently more resistant to sunlight inactivation than were FC. Finally, evidence was obtained to show that sunlight initially stresses the bacteria but eventually causes cell death.     

Fecal indicator bacteria persistence under natural conditions in an ice-covered river.

Total coliform (TC), fecal coliform (FC), and fecal streptococcus (FS) survival characteristics, under natural conditions at 0 degrees C in an ice-covered river, were examined during February and March 1975. The membrane filter (MF) technique was used throughout the study, and the multiple-tube (MPN) method was used in parallel on three preselected days for comparative recovery of these bacteria. Survival was studied at seven sample stations downstream from all domestic pollution sources in a 317-km reach of the river having 7.1 days mean flow time (range of 6.0 to 9.1 days). The mean indicator bacteria densities decreased continuously at successive stations in this reach and, after adjustment for dilution, the most rapid die-off was found to occur during the first 1.9 days, followed by a slower decrease. After 7.1 days, the relative survival was TC less than FC less than FS, with 8.4%, 15.7%, and 32.8% of the initial populations remaining viable, respectively. These rates are higher than previously reported and suggest that the highest survival rates for these bacteria in receiving streams can be expected at 0 degree C under ice cover. Additionally, the FC-FS ratio was greater than 5 at all stations, indicating that this ratio may be useable for determining the source of fecal pollution in receiving streams for greater than 7 days flow time at low water temperatures. The MPN and MF methods gave comparable results for the TC and FS at all seven sample stations, with both the direct and verified MF counts within the 95% confidence limits of the respective MPNs in most samples, but generally lower than the MPN index. Although FC recovery on membrane filters was comparable results at stations near the pollution source. However, the results became more comparable with increasing flow time. The results of this study indicate that heat shock is a major factor in suppression of the FC counts on the membrane filters at 44.5 degree C. Heat shock may be minimized by extended incubation at 35 degrees C before exposure to the higher temperature.     

Evaluation of the 7-h membrane filter test for quantitation of fecal coliforms in water.

The 7-h fecal coliform (FC) test for detection of FC organisms in water was evaluated to establish its validity and usefulness for emergency and disaster situations. The waters tested consisted of routine samples collected for public health surveillance and enforcement purposes. A total of 984 water samples from throughout California were assayed. These included samples from coastal salt waters, rivers, canals, and reservoirs, in addition to potable and miscellaneous freshwater sources. A portion of each sample was tested concurrently by both the 7-h FC test and the most-probable-number FC five-tube test. The 7-h FC test samples were incubated for 7 to 7.25 h at 41.5 degrees C. Overall, greater than 90% agreement was obtained between the methods in determining whether the water quality was acceptable or unacceptable. Statistical analysis of the 984 samples confirmed that the 7-h FC method was a suitable alternative to the most-probable-number FC method for evaluation of freshwater samples. During emergencies or disasters, the 7-h FC test could provide a means for detection of fecal contamination of water with results available in less than 1 day.     

Evaluation of the 7-h membrane filter test for quantitation of fecal coliforms in water

The 7-h fecal coliform (FC) test for detection of FC organisms in water was evaluated to establish its validity and usefulness for emergency and disaster situations. The waters tested consisted of routine samples collected for public health surveillance and enforcement purposes. A total of 984 water samples from throughout California were assayed. These included samples from coastal salt waters, rivers, canals, and reservoirs, in addition to potable and miscellaneous freshwater sources. A portion of each sample was tested concurrently by both the 7-h FC test and the most-probable-number FC five-tube test. The 7-h FC test samples were incubated for 7 to 7.25 h at 41.5 degrees C. Overall, greater than 90% agreement was obtained between the methods in determining whether the water quality was acceptable or unacceptable. Statistical analysis of the 984 samples confirmed that the 7-h FC method was a suitable alternative to the most-probable-number FC method for evaluation of freshwater samples. During emergencies or disasters, the 7-h FC test could provide a means for detection of fecal contamination of water with results available in less than 1 day.     

A simple bioluminescence procedure for early warning detection of coliform bacteria in drinking water

Traditional cultivation-dependent tests for coliform bacteria in food and drinking water take 18–24 h to complete. Bioluminescence-based enzyme assays can potentially reduce analysis time for indicator bacteria such as coliforms. In the present study, we developed a simple presence/absence (P/A) bioluminescence procedure for rapid detection of coliform bacteria in groundwater-based drinking water. The bioluminescence procedure targeting β-d-galactosidase activity in coliform bacteria was based on hydrolysis of 6-O-β-galactopyranosyl-luciferin. Bacteria immobilized on membrane filters were enriched for 6–8 h in selective media containing isopropyl-β-d-thiogalactopyranoside (IPTG) to induce β-d-galactosidase activity in coliform bacteria. The equivalent of approximately 300 E. coli cells was required for bioluminescence detection of β-d-galactosidase activity. In comparison, PCR based detection of E. coli in drinking water required approximately 30 target cells. Analysis of contaminated drinking water samples showed comparable results for coliform bacteria using traditional multiple-tube fermentation, Colilert-18, and the bioluminescence procedure. Aeromonas hydrophila or indigenous groundwater bacteria did not interfere with the procedure. The bioluminescence procedure can be combined with commercial substrates such as Fluorocult or Colilert-18, and will allow the detection of one coliform in 100 ml drinking water within one working day. The results suggest the bioluminescence assays targeting β-d-galactosidase activity may be used for or for early warning screening of drinking water and/or rapid identification of contaminated drinking water wells.     

Removal and inactivation of bacteria during alum treatment of a lake.

Flocculation and removal of bacteria were observed during two separate aluminum sulfate (alum) treatments for removal of phosphorus from a eutrophic recreational lake. In addition, die-off and release of bacteria from alum floc were studied in columns under laboratory conditions. Membrane filtration and spread plates were used to determine concentrations of indicator species and total cultivatable bacteria, respectively. During the alum treatment of the lake, 90% of the fecal coliform (FC) population and ca. 70% of the fecal streptococci population were removed from the water column within 72 h. Numbers of FC in the floc on the lake bottom exceeded 2,400/100 ml at 120 h compared with the pretreatment concentration of 30 FC/100 ml. Inactivation of FC in the floc proceeded at a rate of 200 FC/100 ml per 24 h. In a second alum application to the lake, 95% of the total culturable bacterial population was removed from the water column. In a laboratory column study of survival and release rates, over 90% of an Escherichia coli suspension was concentrated in a floc formed at the bottom. E. coli was not released from the floc. The numbers of and survival of E. coli in the floc suggest the probable concentration of other enteric organisms, including pathogens. Thus, the floc poses a potential human health risk if ingested by swimmers or if others use the lake as a potable water source.     

Application of rapid enzyme assay techniques for monitoring of microbial water quality

Rapid enzyme assay techniques based on direct measurement of beta-d-galactosidase (GALase) or beta-d-glucuronidase (GLUase) activity without selective cultivation are used for rapid estimation of the level of coliform bacteria and Escherichia coli in water samples. Reported detection limits using fluorogenic substrates correspond to culturable target bacteria concentrations that can be appropriate within present guidelines for recreational waters. The rapidity, that is detection within one hour, compromises the specificity of the assay; enzyme activity contributions from other than target bacteria need to be considered, particularly at low levels of target bacteria. Enzyme activities are more persistent than the culturability of target bacteria to environmental and disinfection stress, thus water samples may express enzyme activities of both culturable and viable non-culturable cells.     

Modulation of the phenotype in dominant erythropoietic protoporphyria by a low expression of the normal ferrochelatase allele.

Erythropoietic protoporphyria (EPP) is a monogenic inherited disorder of the heme biosynthetic pathway due to ferrochelatase (FC) deficiency. EPP is generally considered to be transmitted as an autosomal dominant disease with incomplete penetrance, although autosomal recessive inheritance has been documented at the enzymatic and molecular level in some families. In the dominant form of EPP, statistical analysis of FC activities documented a significantly lower mean value in patients than in asymptomatic carriers, suggesting a more complex mode of inheritance. To account for these findings, we tested a multiallelic inheritance model in one EPP family in which the enzymatic data were compatible with this hypothesis. In this EPP family, the specific FC gene mutation was an exon 10 skipping (delta Ex10), resulting from a G deletion within the exon 10 consensus splice donor site. The segregation of all FC alleles within the family was followed using the delta Ex10 mutation and a new intragenic dimorphism (1520 C/T). mRNAs transcribed from each FC allele were then subjected to relative quantification by a primer extension assay and to absolute quantification by a ribonuclease protection assay. The data support the hypothesis that in this family the EPP phenotype results from the coinheritance of a low output normal FC allele and a mutant delta Ex10 allele.     

Rapid Determination of the Presence of Enteric Bacteria in Water

A rapid and sensitive method is described for the detection of bacteria in water and various other natural substrates by the isolation of specific bacteriophage. By the addition of large numbers of the organism in question to the sample, the presence of virulent bacteriophage can be demonstrated in as little as 6 to 8 h. Fecal coliform, total coliform, and total coliphage counts were determined for over 150 water samples from several geographical areas over a period of 2 years. Computer analysis of the data shows a high degree of correlation between fecal coliforms and the coliphage present in the samples. With a high correlation coefficient between fecal coliform and coliphage counts, predictions of the fecal coliforms may be made by enumeration of the phage.     

Bacterial,nutrients and heavy metal ions pollution assessment along the eastern coastal area of the United Arab Emirates

A program to monitor nutrients and heavy metal ions concentrations in addition to selected bacterial communities counts was carried out at three sites along the eastern coast of the United Arab Emirates on the Gulf of Oman. Total saprophytic bacteria (TSB), salt tolerant saprophytic bacteria (STSB), Gram-negative bacteria (GNB), total coliform (TC), and faecal coliform bacteria (FC) were enumerated. The concentrations of heavy metal ions including Mn, Fe, Pb, Cd, Zn, Cr, Ni, Co and Cu were determined by atomic absorption spectrometry. Bacterial counts had a distinct pattern with peaks in mid spring (May) and in autumn (October). The TSB, STSB and the GNB ranges fluctuated between, 3.3 × 10⁴ to 4 × 10⁵, 1.8 × 10⁴ to 2.5 × 10⁵ and 0.7 × 10⁴ to 1.3 × 10⁵ colony forming units (CFU) ml^–1, respectively. Total and faecal coliform bacteria fluctuated depending on several factors including the presence of nearby recreation and commercial areas, but were at no time consistently high.Bacillus, Pseudomonas, Staphylococcus, Micrococcus and Alteromonas, were the predominant bacterial genera in these waters. Major nutrients and trace heavy metal ions concentrations were within the normal ranges for sea water with occasional sharp fluctuations in some sites. It was generally concluded that the area is slightly polluted.     

The influence of rainfall on the incidence of microbial faecal indicators and the dominant sources of faecal pollution in a Florida river

AIMS: An assessment of microbial densities in an urbanized Florida watershed was performed during a period of changing rainfall patterns to investigate the role of climate coupled with urbanization in declining water quality. METHODS AND RESULTS: Concentrations of traditional and alternative faecal indicators were assessed by standard methods over 24 months. Sources of faecal contamination were determined by antibiotic resistance analysis (ARA) of faecal coliform (FC) bacteria. Composite indices of indicator organisms based on a suite of microbial measurements were used to quantify pollution impacts in the river. ARA results found that FC from wild animal sources dominated during the drought, and the relative frequency of FC from human sources increased after cumulative rainfall increased to near-normal levels. CONCLUSIONS: Changes observed in faecal indicator densities and in FC sources during changing rainfall patterns strongly suggest a role of precipitation on the sources and extent of microbial pollution in urbanized coastal watersheds. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial source tracking coupled with a composite index of microbial contamination resulted in a more complete picture of microbial pollution within the river, as opposed to the general practice of reliance on one indicator organism. Improved land use decisions in urban areas are necessary to insure maintenance of coastal environmental health under changing climate patterns and population density.     

Detection and enumeration of coliforms in drinking water: current methods and emerging approaches.

The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments.Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water.The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes beta-D galactosidase and beta-D glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold.Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (beta-galactosidase gene) and the uidA gene (beta-D glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort.This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.     

Blood serum concentration of lipids and lipoproteins and body composition

The purpose of this study is the analysis of the relationship of blood serum apolipoprotein E (apoE), total cholesterol (TC), triglycerides (TG) and high-density and low-density lipoproteins (HDL, LDL), with body mass index (BMI), relative body surface area (RBSA) and body muscle (BM) and body fat (BF). The subjects are males and females aged 14-16 (adolescent age group 1: n1M = 141, n1F = 151) and 18-25 (young adult group 2, n2M = 16, n2F = 46). Significant correlations of serum TG and HDL with somatometric indicators were not observed. In the female samples, TC content directly correlates (p < 0.05) with BF (r1 = 0.164; r2 = 0.418) and negatively correlates with BM (r1 = -0.165; r2 = -0.352). The blood serum concentration of apoE is significantly correlated with body composition in adolescent females (for BF r1 = -0.168; for BM r1 = 0.266; p < 0.05); in males 14-16 years old, the both correlations have a significance level p < 0.06. In young adult females TC and LDL content negatively correlates with RBSA (r2 = -0.386 and -0.377 respectively; p < 0.05) and positively correlates with BMI (r2 = 0.413 and 0.415 respectively; p < 0.05). Adolescent females and young adult females have opposite relationships between FC and apoE concentration. In females 14-15 years old apoE concentration decreases as FC increases. In females 16-17 the correlation disappears, and in older females apoE concentration and FC increase together.