MI Agar Research Articles

Novel methods for global water safety monitoring: comparative analysis of low-cost, field-ready E. coli assays

Current microbiological water safety testing methods are not feasible in many settings because of laboratory, cost, and other constraints, particularly in low-income countries where water quality monitoring is most needed to protect public health. We evaluated two promising E. coli methods that may have potential in at-scale global water quality monitoring: a modified membrane filtration test followed by incubation on pre-prepared plates with dehydrated culture medium (CompactDryTM), and 10 and 100 ml presence–absence tests using the open-source Aquatest medium (AT). We compared results to membrane filtration followed by incubation on MI agar as the standard test. We tested 315 samples in triplicate of drinking water in Bangalore, India, where E. coli counts by the standard method ranged from non-detect in 100 ml samples to TNTC (>200). Results suggest high sensitivity and specificity for E. coli detection of candidate tests compared with the standard method: sensitivity and specificity of the 100 ml AT test was 97% and 96% when incubated for 24 h at standard temperature and 97% and 97% when incubated 48 h at ambient temperatures (mean: 27 °C). Sensitivity and specificity of the CompactDryTM test was >99 and 97% when incubated for 24 h at standard temperature and >99 and 97% when incubated 48 h at ambient temperatures. Good agreement between these candidate tests compared with the reference method suggests they are suitable for E. coli monitoring to indicate water safety.

A novel molecular quantitative method for rapid and sensitive detection ofEscherichia colifrom roof-harvested rainwater

Comparison of the sensitivity and specificity of CoDEX-qPCR with that of the U.S. EPA MI agar method 1604 anduidA,yaiOandtufgene-based qPCR.

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.

Draft Genome Sequence of Raoultella planticola, Isolated from River Water

We isolated Raoultella planticola from a river water sample, which was phenotypically indistinguishable from Escherichia coli on MI agar. The genome sequence of R. planticola was determined to gain information about its metabolic functions contributing to its false positive appearance of E. coli on MI agar. We report the first whole genome sequence of Raoultella planticola.

Draft Genome Sequence of Buttiauxella agrestis, Isolated from Surface Water

MI agar is routinely used for quantifying Escherichia coli in drinking water. A suspect E. coli colony isolated from a water sample was identified as Buttiauxella agrestis. The whole genome sequence of B. agrestis was determined to understand the genetic basis for its phenotypic resemblance to E. coli on MI agar.

Comparison of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms in water.

The MI agar, Colilert(®), Chromocult coliform(®) agar, and DC with BCIG agar chromogenic culture-based methods used to assess microbiological quality of drinking water were compared in terms of their ubiquity, sensitivity, ease of use, growth of atypical colonies and affordability. For ubiquity, 129 total coliform (representing 76 species) and 19 Escherichia coli strains were tested. Then, 635 1-L well water samples were divided into 100 mL subsamples for testing by all four methods. Test results showed that 70.5, 52.7, 36.4, and 23.3% of the non-E. coli total coliform strains and 94.7, 94.7, 89.5, and 89.5% of the 19 E. coli strains yielded a positive signal with the four methods, respectively. They also yielded a total coliform positive signal for 66.5, 51.7, 64.9, and 55.0% and an E. coli positive signal for 16.1, 14.8, 17.3, and 13.4% of the 635 well water samples tested, respectively. Results showed that Colilert(®) is the most expensive method tested in terms of reactants, yet it is the easiest to use. Large numbers of atypical colonies were also often observed on Chromocult coliform(®) and DC with BCIG, thereby challenging the target microorganism count. Thus, the MI agar method seems to be the best option for the assessment of drinking water quality.

Molecular method for detection of total coliforms in drinking water samples.

This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.

Detection of Escherichia coli colonies on confluent plates of chromogenic media used in membrane filtration

MI agar (MI), Chromocult® Coliform agar ES (Chromocult), and DC with BCIG agar (DC) are chromogenic membrane filtration culture-based methods used to assess microbiological water quality. In this study, their ability to detect Escherichia coli colonies on confluent growth plates was determined by testing water samples containing increasing concentrations of a non-E. coli growing bacterium, Citrobacter youngae. Then, their ability to inhibit the growth of non-coliform bacteria was determined by simultaneously testing 603 well water samples. Results were compared with those obtained with mFC and Colilert® methods. Results showed that the E. coli count was difficult to determine on mFC, Chromocult and DC when non-E. coli colonies reached levels of 104colony forming units (CFU)/100mL. However, the E. coli count did not interfere with Colilert until non-E. coli colonies reached concentrations of 107CFU/100mL. No inhibition was observed with MI as E. coli colonies could be easily detected in the presence of at least 107CFU/100mL of C. youngae. Using well water samples, confluent growth plates were observed for 144, 177, and 185 of the 603 well water samples tested with the MI, Chromocult and DC methods, respectively. Among these confluent growth plates, E. coli colonies were not detected for 10, 20, and 31 water samples. However, they were detected by the mFC and/or Colilert methods. Thus, among the three methods tested, the MI method presented the lowest grow rate of atypic colonies and was the only one that presents no interference in the E. coli count.

Comparative analysis of classical and molecular microbiology methods for the detection of Escherichia coli and Enterococcus spp. in well water

The microbiological quality of 165 1 litre well water samples collected in the Québec City region was assessed by culture-based methods (mFC agar, Chromocult coliform agar, Colilert(®), MI agar, Chromocult enterococci, Enterolert™, and mEI agar) and by a molecular microbiology strategy, dubbed CRENAME-rtPCR, developed for the detection of Escherichia coli, Enterococcus spp., Enterococcus faecalis/faecium, and Bacillus atrophaeus subsp. globigii. In these drinking water samples, approved culture-based methods detected E. coli at rates varying from 1.8 to 3.6% and Enterococcus spp. at rates varying from 3.0 to 11.5%, while the molecular microbiology approach for E. coli was found to be as efficient, detecting contamination in 3.0% of samples. In contrast, CRENAME-rtPCR detected Enterococcus spp. in 27.9% of samples while the E. faecalis/faecium molecular assay did not uncover a single contaminated sample, thereby revealing a discrepancy in the coverage of waterborne enterococcal species detected by classical and molecular microbiology methods. The validation of the CRENAME-E. coli rtPCR test as a new tool to assess the quality of drinking water will require larger scale studies elaborated to demonstrate its equivalence to approved methods.

False-positive identification ofEscherichia coliin treated municipal wastewater and wastewater-irrigated soils

The increasing use of treated wastewater for irrigation heightens the importance of accurate monitoring of water quality. Chromogenic media, because they are easy to use and provide rapid results, are often used for detection of Escherichia coli in environmental samples, but unique levels of organic and inorganic compounds alter the chemistry of treated wastewater, potentially hindering the accurate performance of chromogenic media. We used MI agar and molecular confirmatory methods to assess false-positive identification of E. coli in treated wastewater samples collected from municipal utilities, an irrigation holding pond, irrigated soils, and in samples collected from storm flows destined for groundwater recharge. False-positive rates in storm flows (4.0%) agreed closely with USEPA technical literature but were higher in samples from the pond, soils, and treatment facilities (33.3%, 38.0%, and 48.8%, respectively). Sequencing of false-positive isolates confirmed that most were, like E. coli, of the family Enterobacteriaceae, and many of the false-positive isolates were reported to produce the β-d-glucuronidase enzyme targeted by MI agar. False-positive identification rates were inversely related to air temperature, suggesting that seasonal variations in water quality influence E. coli identification. Knowledge of factors contributing to failure of chromogenic media will lead to manufacturer enhancements in media quality and performance and will ultimately increase the accuracy of future water quality monitoring programs.

False-negative β-d-glucuronidase reactions in membrane lactose glucuronide agar medium used for the simultaneous detection of coliforms andEscherichia colifrom water

Testing for beta-D-glucuronidase activity has become the basis of many methods for the detection of Escherichia coli in both food and water. Used in combination with tests for the presence of beta-D-glucuronidase, these tests offer a simple method for simultaneously detecting coliforms and E. coli. The purpose of this study was to determine the effectiveness of several different procedures in detecting beta-D-glucuronidase activity and hence in detecting E. coli. The ability of membrane lactose glucuronide agar (MLGA), Colilert-18, MI agar, Colitag and Chromocult agar to detect beta-D-glucuronidase activity was tested with over 1000 isolates of E. coli recovered from naturally contaminated water samples. Four of the media gave very similar results but MLGA failed to detect glucuronidase activity in 15.6% of the cultures tested. MLGA had very poor sensitivity for the detection of beta-D-glucuronidase activity in strains of E. coli isolated from naturally contaminated water. This is probably because of the fact that beta-D-glucuronidase activity is pH-sensitive and that acid is formed by E. coli during fermentation of lactose in MLGA. The detection of E. coli in drinking water is the primary test used to establish faecal contamination. The poor sensitivity of MLGA in detecting beta-D-glucuronidase activity suggests that this medium and others containing high concentrations of fermentable carbohydrate should not be used for the detection of E. coli.

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.

Evaluation of chromogenic technologies for use in Australian potable water

To compare the use of MI agar, Membrane Lactose Glucoronide Agar (MLGA), CM1046 agar and Colilert-18 (Defined Substrate Technology, IDEXX Laboratories Pty. Ltd., Sydney) on Australian potable water. Both potable (n = 369) and nonpotable waters (n = 35) were analysed by membrane filtration using chromogenic agars as well as Colilert-18 over a period of 12 months. Recoveries of stressed organisms on these chromogenic media were also investigated. Agar-based chromogenic technologies compared favourably to Colilert-18 for chlorinated waters, but there are possible limitations when using these agars for chloraminated waters. Additionally, the breakthrough of problematic organisms, especially oxidase positive organisms, may lead to misrepresentation or over-estimation of E. coli and total coliforms, particularly on MLGA and CM1046. The recovery of stressed organisms was favoured in the Colilert-18 system when compared to chromogenic agars. MI agar performed better than the other chromogenic agars with respect to recovery and colour identification and discrimination of organisms, and compared favourably with Colilert-18. The use of chromogenic agars in chloraminated waters should be done cautiously. This study provides comparison data for laboratories looking to adopt chromogenic technologies, and is especially important for Australian laboratories wanting to uptake the use of MI agar (as used in USEPA method 1604) for routine use and for gaining accreditation. Additionally, to the best of our knowledge, this is the first reported evaluation of these agars in chloraminated waters and is especially timely as the use of this disinfection agent is increasing.

Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modified chromogenic agar.

We validated a novel method for screening Escherichia coli resistance to antibiotics in environmental samples using modified Difco MI agar (Becton Dickinson) impregnated with selected antibiotics (tetracycline, ampicillin, cephalexin, and sulfamethoxazole), termed MI-R. This method combines an existing rapid assessment technique for E. coli enumeration with clinical reference data for breakpoint analysis of antibiotic resistance and was developed to address issues encountered when clinical methods are used with environmental samples. Initial trials conducted using strains of E. coli with resistance to the selected antibiotics showed that this method was reproducible and accurate with respect to antibiotic resistance. Trials using wastewater effluent demonstrated the precision of the method, and the levels of resistance found in effluent were directly comparable to the levels of antibiotic resistance determined using the more traditional CLSI (formerly NCCLS) disk susceptibility test. All wastewater isolates growing on MI-R plates were confirmed to be resistant using the CLSI disk susceptibility test. Bacterial resistance to ampicillin (38% +/- 4% overall), sulfamethoxazole, tetracycline (21% +/- 3% overall), and ciprofloxacin (6% +/- 1%) were found in wastewater effluent. A successful trial was also conducted with water collected from the Brisbane River, Australia. The levels of antibiotic resistance in E. coli ranged from 0 to 47% for ampicillin, from 0 to 24% for tetracycline, from 0 to 63% for sulfamethoxazole, and from 0 to 1% for ciprofloxacin, with the highest incidence of resistance associated with wastewater treatment plant discharges. This method has great potential for rapid and representative assessment of antibiotic resistance in E. coli and could allow increased sample analysis, resulting in greater confidence in spatial analysis in environmental studies.

Comparison of methods for determining Escherichia coli concentrations in recreational waters

Seventy water samples were collected from three Lake Erie beaches to compare recoveries of Escherichia coli ( E. coli) using the USEPA-recommended method for recreational waters (mTEC) to recoveries using three alternative methods (MI, modified mTEC, and Colilert). Statistical tests showed no differences in recoveries of E. coli between MI and mTEC; however, statistically-significant differences were found between modified mTEC or Colilert and mTEC. The MI agar method provided the most similar assessment of recreational water quality to mTEC among the three alternative methods tested. The range of differences between Colilert and mTEC was widest among the three alternative methods. In a sample group with a range of values near the single-sample bathing-water standard, recoveries of E. coli were statistically lower using modified mTEC than mTEC; however, MI and Colilert compared well to mTEC in this range. Because samples were collected in a small geographic area, more work is necessary to test within-method variability of the modified mTEC, MI, and Colilert methods and to evaluate these methods as substitutes for the mTEC method in a variety of recreational waters.

Interlaboratory evaluation of MI agar and the US Environmental Protection Agency-Approved Membrane Filter Method for the recovery of total coliforms and Escherichia coli from drinking water

A new membrane filter (MF) medium, MI agar, recently validated for use in recovering chlorine-damaged total coliforms (TC) and Escherichia coli from drinking water, was compared to the US Environmental Protection Agency (EPA)-approved MF method (mEndo agar and nutrient agar supplemented with 4-methylumbelliferyl-β- d-glucuronide) in a collaborative study. Six wastewater-spiked Cincinnati tap water samples, containing three different concentrations of E. coli (≤ 10, 11–30, and >30 E. coli/100 ml), were analyzed concurrently by 19 geographically dispersed laboratories. Results showed that although the overall recoveries of TC and E. coli with MI agar were 12% and 26% greater, respectively, than those of the approved method, the differences were not statistically significant (α=0.05). However, the overall recovery of background/non-coliforms by the new method, 5% of the mEndo agar recovery, was significantly lower ( P=0.0009). Since the results of this study show MI agar to be equal to or better than the current method, it meets the criteria for routine compliance monitoring of drinking water.

New medium for the simultaneous detection of total coliforms and Escherichia coli in water

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.