Since 2002, the United States Environmental Protection Agency (USEPA) has approved ten enzyme-based total coliform and E. coli detection tests for examination of drinking water. These tests include: Colilert, Colilert-18, Colisure, m-Coli Blue 24, Readycult Coliforms 100, Chromocult, Coliscan, E * Colite, Colitag and MI Agar. The utility of the enzyme based test systems is based on both the ability of the test to detect the target organisms at low levels and the ability of the test system to suppress the growth of non-target organisms that might result in false positive results. Differences in the ability of some of these methods to detect total coliform and E. coli, as well as suppress Aeromonas spp., a common cause of "false positive" results, have been observed. As a result, this study was undertaken to elucidate the strengths and weaknesses of each method. Water samples were collected from three geographically and chemically diverse groundwaters in Wisconsin. One-hundred milliliter aliquots were individually spiked with both low concentrations (one to ten organisms) and high concentrations (fifty to one-hundred) of each of five different total coliform organisms (Serratia, Citrobacter, Enterobacter, E. coli, & Klebsiella). These spiked samples were used to test the capability of ten enzyme-based test systems to both detect and enumerate the spiked organisms. In addition, 100 ml samples were independently spiked with two different strains of Aeromonas spp. at six different levels, to assess the ability of each enzyme-based test to suppress Aeromonas spp. Analysis of the data indicated that wide variability exists among USEPA approved tests to detect and quantify total coliforms, as well as suppress Aeromonas spp.

Gastrointestinal infections of Aeromonas species are generally considered waterborne; for this reason, Aeromonas hydrophila has been placed on the United States Environmental Protection Agency Contaminant Candidate List of emerging pathogens in drinking water. In this study, we compared pulsed-field gel electrophoresis patterns of Aeromonas isolates from stool specimens of patients with diarrhea with Aeromonas isolates from patients’ drinking water. Among 2,565 diarrheic stool specimens submitted to a Wisconsin clinical reference laboratory, 17 (0.66%) tested positive for Aeromonas . Groundwater isolates of Aeromonas were obtained from private wells throughout Wisconsin and the drinking water of Aeromonas -positive patients. The analysis showed that the stool and drinking water isolates were genetically unrelated, suggesting that in this population Aeromonas gastrointestinal infections were not linked with groundwater exposures.

Escherichia coli is a routinely used microbiological indicator of water quality. To determine whether holding time and storage conditions had an effect on E. coli densities in surface water, studies were conducted in three phases, encompassing 24 sites across the United States and four commonly used monitoring methods. During all three phases of the study, E. coli samples were analyzed at time 0 and at 8, 24, 30, and 48 h after sample collection. During phase 1, when 4°C samples were evaluated by Colilert or by placing a membrane onto mFC medium followed by transfer to nutrient agar containing 4-methylumbelliferyl-␤-D-glucuronide (mFC/NA-MUG), three of four sites showed no significant differences throughout the 48-h study. During phase 2, five of seven sites showed no significant difference between time 0 and 24 h by membrane filtration (mFC/NA-MUG). When evaluated by the Colilert method, five of seven sites showed no significant difference in E. coli density between time 0 and 48 h. During phase 3, 8 of 13 sites showed no significant differences in E. coli densities between time 0 and the 48-h holding time, regardless of method. Based on the results of these studies, it appears that if samples are held below 10°C and are not allowed to freeze, most surface water E. coli samples analyzed by commonly used methods beyond 8 h after sample collection can generate E. coli data comparable to those generated within 8 h of sample collection. Notwithstanding this conclusion, E. coli samples collected from surface waters should always be analyzed as soon as possible.Escherichia coli testing is an important tool used by public health experts for the prevention of waterborne disease. The detection of E. coli in a water sample from an environmental source provides direct evidence of fecal contamination. Regulatory agencies are increasingly requiring more emphasis on E. coli testing as part of programs aimed at curtailing waterborne disease. Holding time and temperature can have a significant impact on the density of microbiological indicators at the time of sample analysis (4, 5, 7). Recommendations for E. coli holding times range from 8 h (2, 3, 9) to 24 h (8), and holding temperatures below 10°C are generally considered acceptable (2,3,8,9). It is also recommended that when transport conditions result in delays longer than 6 h, the use of field laboratory facilities located at the site of collection or delayed incubation procedures be considered (2). The Surface Water Treatment Rule requirements of the U.S. Environmental Protection Agency (USEPA) for total coliform and fecal coliform monitoring of surface water used as drinking water sources (3) specify that the time from sample collection to initiation of analysis is not to exceed 8 h; the regulations also encourage (but do not require) drinking water system personnel to hold samples at below 10°C during transit.Unfortunately, data from evaluations of microbiological indicator density that support current holding time recommendations are limited, particularly for E. coli....

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10 6 CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.A scientific breakthrough occurred in 1982 when J. R. Warren and B. Marshall isolated a bacterium and showed that it causes gastritis and stomach ulcers that affect millions of humans worldwide (10,12). Today this etiology has been proven to the extent that the National Institutes of Health recommends treatment with antibiotics for all patients with peptic ulcers, which are almost exclusively attributed to infection with the bacterium Helicobacter pylori (4). The scope of gastric illnesses around the world is vast, and in the United States alone, over 5,000,000 people are diagnosed annually with ulcers, 1,000,000 people are hospitalized, 40,000 people undergo surgery, and 6,500 people die from ulcer-related complications (11,21). Estimates suggest that as many as 50% of adult Americans carry the pathogen, most asymptomatically, and in lessdeveloped countries human carriers represent up to 90% of the populations (15).The source of human infection is not yet known, and until recently, the natural reservoir for H. pylori was thought to be the human gastrointestinal tract (1). However, the association of Helicobacter with nonhuman sources, such as livestock (23), domestic cats, (17), and vegetables (6), prompted researchers to look at en...

The goal of this project was to evaluate the removal of emerging pathogens and pathogen indicators by pilot-scale coagulation, flocculation, sedimentation, and granular media filtration. The emerging pathogens included Cryptosporidium parvum oocysts, Encephalitozoon intestinalis spores, Escherichia coli O157:H7, and Aeromonas hydrophila. Bacteriophage MS2 and turbidity were used as pathogen indicators. This work revealed that some emerging pathogens were removed much more effectively than others. A. hydrophila was removed more effectively than C. parvum, and E. intestinalis spores and E. coli O157:H7 were the least effectively removed. For the water tested in this study, the results suggest that a change in filter effluent turbidity requirements from 0.5 to 0.3 ntu would not achieve a significant improvement in the reliability of pathogen removal. However, by setting filter effluent turbidity goals below 0.2 ntu, significant improvements in microbiological quality could be obtained.In general, the pilot-plant data suggest that good removal of both turbidity and natural organic matter (NOM) decreases the risk of achieving poor emerging pathogen removal. In other words, optimizing the coagulation process for reduction of turbidity and NOM would improve removal of emerging pathogens.