Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert^® (Colilert), Readycult^® Coliforms 100 (Readycult), Chromocult^® Coliform agar ES (Chromocult), and MI agar (MI) are β-galactosidase and β-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect β-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect β-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected β-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected β-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect β-glucuronidase production and MI the weakest to detect β-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods.     

Analytical limits of four beta-glucuronidase and beta-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert (Colilert), Readycult Coliforms 100 (Readycult), Chromocult Coliform agar ES (Chromocult), and MI agar (MI) are beta-galactosidase and beta-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect beta-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect beta-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect beta-glucuronidase production and MI the weakest to detect beta-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.     

Enumeration of Escherichia coli and coliforms in surface water by multiple tube fermentation and membrane filter methods

The current investigation was carried out in order to compare directly the multiple tube fermentation method (MTF), using standard procedures (lactose broth, LB) and the Colilert reagent, with the membrane filter method (MF) using Les Endo agar (LEA), m-faecal coliform agar (mFCA) and chromogenic coliform agar (CCA), for recovery of coliforms and Escherichia coli in 80 surface water samples. Total coliforms were isolated from 100% of samples by all methodologies. Faecal coliforms/E. coli were detected in 100% of samples by MTF methods, but only in 75.5% by MF-mFCA and in 86.2% by MF-CCA. Even if MTF-LB counts were consistently higher, the Colilert reagent accurately determined total coliforms and E. coli levels within 24 h with no additional confirmatory tests. Therefore, it could be a powerful tool for rapidly assessing possible faecal contamination and a suitable alternative to the traditional MTF and MF techniques utilized for coliform detection.     

Comparison of Membrane Filtration and Multiple-Tube Fermentation by the Colilert and Enterolert Methods for Detection of Waterborne Coliform Bacteria, Escherichia coli, and Enterococci Used in Drinking and Bathing Water Quality Monitoring in Southern Sweden

A total of 338 water samples, 261 drinking water samples and 77 bathing water samples, obtained for routine testing were analyzed in duplicate by Swedish standard methods using multiple-tube fermentation or membrane filtration and by the Colilert and/or Enterolert methods. Water samples came from a wide variety of sources in southern Sweden (Skåne). The Colilert method was found to be more sensitive than Swedish standard methods for detecting coliform bacteria and of equal sensitivity for detecting Escherichia coli when all drinking water samples were grouped together. Based on these results, Swedac, the Swedish laboratory accreditation body, approved for the first time in Sweden use of the Colilert method at this laboratory for the analysis of all water sources not falling under public water regulations (A-krav). The coliform detection study of bathing water yielded anomalous results due to confirmation difficulties. E. coli detection in bathing water was similar by both the Colilert and Swedish standard methods as was fecal streptococcus and enterococcus detection by both the Enterolert and Swedish standard methods.     

Occurrence of Microbial Indicators and Clostridium perfringens in Wastewater, Water Column Samples, Sediments, Drinking Water, and Weddell Seal Feces Collected at McMurdo Station, Antarctica

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.     

Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology

Many water utilities are required to monitor source water for the presence of total coliforms, fecal coliforms, or both. The Colilert system, an application of the defined substrate technology, simultaneously detects the presence of both total coliforms and Escherichia coli directly from a water sample. After incubation, the formula becomes yellow if total coliforms are present and fluorescent at 366 nm if E. coli is in the same sample. No confirmatory tests are required. The Colilert system was previously assessed with distribution water in a national evaluation in both most-probably-number and presence-absence formats and found to produce data equivalent to those obtained by using Standard Methods for the Examination of Water and Wastewater (Standard Methods). The Colilert system was now compared with Standard Methods multiple-tube fermentation (MTF) for the enumeration of total coliforms and E. coli from surface water. All MTF tubes were confirmed according to Standard Methods, and subcultures were made to identify isolates to the species level. The Colilert system was found equally sensitive to MTF testing by regression, t test, chi-square, and likelihood fraction analyses. Specificity of the Colilert system was shown by the isolation of a species of total coliform or E. coli after the appropriate color change. The Colilert test can be used for source water samples when enumeration is required, and the benefits previously described for distribution water testing--sensitivity, specificity, less labor, lower cost, faster results, no noncoliform heterotroph interference--are applicable to this type of water analysis.     

Enumeration of total coliforms and Escherichia coli from source water by the defined substrate technology.

Many water utilities are required to monitor source water for the presence of total coliforms, fecal coliforms, or both. The Colilert system, an application of the defined substrate technology, simultaneously detects the presence of both total coliforms and Escherichia coli directly from a water sample. After incubation, the formula becomes yellow if total coliforms are present and fluorescent at 366 nm if E. coli is in the same sample. No confirmatory tests are required. The Colilert system was previously assessed with distribution water in a national evaluation in both most-probably-number and presence-absence formats and found to produce data equivalent to those obtained by using Standard Methods for the Examination of Water and Wastewater (Standard Methods). The Colilert system was now compared with Standard Methods multiple-tube fermentation (MTF) for the enumeration of total coliforms and E. coli from surface water. All MTF tubes were confirmed according to Standard Methods, and subcultures were made to identify isolates to the species level. The Colilert system was found equally sensitive to MTF testing by regression, t test, chi-square, and likelihood fraction analyses. Specificity of the Colilert system was shown by the isolation of a species of total coliform or E. coli after the appropriate color change. The Colilert test can be used for source water samples when enumeration is required, and the benefits previously described for distribution water testing--sensitivity, specificity, less labor, lower cost, faster results, no noncoliform heterotroph interference--are applicable to this type of water analysis.     

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods.

Three commercially available kits that were supplemented with substrates for enzyme reactions were evaluated to determine their abilities to detect coliforms and fecal coliforms in foods. Japanese and U.S. Food and Drug Administration standard methods, as well as two agar plate methods, were compared with the three commercial kits. A total of 50 food samples from various retailers were examined. The levels of detection of coliforms were high with the commercial kits (78 to 98%) compared with the levels of detection with the standard methods (80 to 83%) and the agar plate methods (56 to 83%). Among the kits tested, the Colilert kit had highest level of recovery of coliforms (98%), and the level of recovery of Escherichia coli as determined by beta-glucuronidase activity with the Colilert kit (83%) was comparable to the level of recovery obtained by the U.S. Food and Drug Administration method (87%). Isolation of E. coli on the basis of the beta-glucuronidase enzyme reaction was found to be good. Levine's eosine methylene blue agar, which has been widely used in various laboratories to isolate E. coli was compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG)-supplemented agar for isolation of E. coli. Only 47% of the E. coli was detected when eosine methylene blue agar was used; however, when violet red bile (VRB)-MUG agar was used, the E. coli detection rate was twice as high. Of the 200 E. coli strains isolated, only 2 were found to be MUG negative, and the gene responsible for beta-glucuronidase activity (uidA gene) was detected by the PCR method in these 2 strains. Of the 90 false-positive strains isolated that exhibited various E. coli characteristic features, only 2 non-E.coli strains hydrolyzed MUG and produced fluorescent substrate in VRB-MUG agar. However, the PCR did not amplify uidA gene products in these VRB-MUG fluorescence-positive strains.     

Total coliform detection in drinking water: comparison of membrane filtration with Colilert and Coliquik.

The Colilert (CL) and Coliquik (CQ) systems were compared in a presence-absence format against the Standard Methods membrane filtration (MF) technique to determine whether differences existed in total coliform detection. Approximately 750 water samples were collected from distribution systems, covered and uncovered storage reservoirs, well sites, and the influent to drinking water treatment plants. Samples were analyzed for total coliforms and heterotrophic bacteria with MF, CL, and CQ. The agreements between CL and MF and between CQ and MF were both greater than 94.8%, which indicates that both may be acceptable methods for total coliform detection. Disagreement between the CL and CQ methods was primarily due to false-negative results. Furthermore, laboratory and field inoculation methods were compared for CL, more than 98% agreement was obtained. This finding indicates that sampling and immediate field inoculation may be an alternative to the traditional laboratory inoculation.     

Total coliform detection in drinking water: comparison of membrane filtration with Colilert and Coliquik

The Colilert (CL) and Coliquik (CQ) systems were compared in a presence-absence format against the Standard Methods membrane filtration (MF) technique to determine whether differences existed in total coliform detection. Approximately 750 water samples were collected from distribution systems, covered and uncovered storage reservoirs, well sites, and the influent to drinking water treatment plants. Samples were analyzed for total coliforms and heterotrophic bacteria with MF, CL, and CQ. The agreements between CL and MF and between CQ and MF were both greater than 94.8%, which indicates that both may be acceptable methods for total coliform detection. Disagreement between the CL and CQ methods was primarily due to false-negative results. Furthermore, laboratory and field inoculation methods were compared for CL, more than 98% agreement was obtained. This finding indicates that sampling and immediate field inoculation may be an alternative to the traditional laboratory inoculation.     

Failure of the most-probable-number technique to detect coliforms in drinking water and raw water supplies.

A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.     

Occurrence of Coliforms, Fecal Coliforms, and Streptococci on Vegetation and Insects

This study considers the sanitary significance of coliforms, fecal coliforms, and streptococci isolated from 152 species of plants and 40 samples of insects. These specimens were collected from various ecological environments and grouped into several categories. Results indicate that typical coliforms of the warm-blooded animal gut contribute a relatively small percentage of the organisms associated with vegetation (14.1%) and insects, (14.9%). A total of 1,203 coliform strains from vegetation and 1,084 coliform strains from insects were classified as to IMViC type and fecal coliform. No type was predominant in either the vegetation or insect groupings. The biochemical results for 646 streptococci from vegetation and 226 cultures from insects were reported. The predominant group, Streptococcus fecalis, as defined by Sherman criteria, constituted a majority of all strains from vegetation and insects. The “Completed Coliform Test” is recommended for the examination of plant and insect specimens to eliminate the many anaerobic and aerobic sporeforming bacteria that frequently produce false positive reactions by the “Confirmed Test” procedure. These findings support the current interpretation of the significance of the fecal coliform test for stream investigations or for surface water quality evaluations.     

Cultivation-independent analysis of bacteria in IDEXX Quanti-Tray/2000 fecal indicator assays

Monitoring microbiological water quality is important for protecting water resources and the health of swimmers. Routine monitoring relies on cultivating fecal indicator bacteria (FIB), frequently using defined substrate technology. Defined substrate technology is designed to specifically enrich for FIB, but a complete understanding of the assay microbiology requires culture-independent analysis of the enrichments. This study aimed to identify bacteria in positive wells of Colilert and Enterolert Quanti-Tray/2000 (IDEXX Laboratories) FIB assays in environmental water samples and to quantify the degree of false-positive results for samples from an urban creek by molecular methods. Pooled Escherichia coli- and Enterococcus-positive Quanti-Tray/2000 enrichments, either from urban creek dry weather flow or municipal sewage, harbored diverse bacterial populations based on 16S rRNA gene sequences and terminal restriction fragment length polymorphism analyses. Target taxa (coliforms or enterococci) and nontarget taxa (Vibrio spp., Shewanella spp., Bacteroidetes, and Clostridium spp.) were identified in pooled and individual positive Colilert and Enterolert wells based on terminal restriction fragments that were in common with those generated in silico from clone sequences. False-positive rates of between 4 and 23% occurred for the urban creek samples, based on the absence of target terminal restriction fragments in individual positive wells. This study suggests that increased selective inhibition of nontarget bacteria could improve the accuracy of the Colilert and Enterolert assays.     

A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)

Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.     

False-positive coliform reaction mediated by Aeromonas in the Colilert defined substrate technology system

The Colilert defined substrate technology system allows specific, one-step detection of both coliforms and Escherichia coli while claiming to suppress the influence of non-coliform heterotrophs. The Colilert assay was examined in order to determine whether organisms from the genus Aeromonas could interfere and cause production of a false-positive coliform result as aquatic Aeromonas are known to constitute a fraction of the heterotrophic population found in drinking water. Results obtained clearly demonstrate that Aeromonas sp. can elicit a positive coliform type reaction at very low densities. Cell suspensions as low as 1 × 10¹ cells 10 ml⁻¹ were observed to yield a positive reaction using Colilert reagent 4 weeks short of shelf-life expiry. Use of aged Colilert for monitoring water quality could lead to over-estimation of coliforms as Aeromonas have been identified in many treated drinking water supplies.     

Evaluation of a new medium for the enumeration of total coliforms and Escherichia coli in Japanese surface waters

Aim: A new medium, EC‐Blue‐10, containing chromogenic and fluorogenic substrates, KNO₃ and sodium pyruvate has been developed for the rapid simultaneous detection and enumeration of total coliforms and Escherichia coli in water. Methods and Results:  Two evaluations of EC‐Blue‐10 were carried out. Firstly, EC‐Blue‐10 was compared with Colilert‐MPN for 96 water samples using MPN for total coliforms and E. coli. Secondly, the detection of coliforms and E. coli were compared using 2400 tubes of EC‐Blue‐10 and Colilert‐MPN. The regression coefficients between EC‐Blue‐10 and Colilert‐MPN for total coliforms and E. coli were 0·91 and 0·89, respectively. For the detection results, the Cohen’s kappa values between the two media were 0·79 for coliforms and 0·72 for E. coli. Conclusions: EC‐Blue‐10 is almost same as Colilert‐MPN for the detection of coliforms and E. coli in surface waters. Further evaluation for EC‐Blue‐10 is needed to verify in different geographical areas. Significance and Impact of the Study: EC‐Blue‐10 is useful method for the rapid and simultaneous detection of total coliforms and E. coli in water sample.     

Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r = 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.