Evaluation of Colilert-18 for Detection of Coliforms and Eschericha coli in Subtropical Freshwater

The accuracy of Colilert-18 as a test for coliforms and Escherichiacoli in subtropical freshwater was evaluated by using API 20Estrips and fatty acid methyl ester analysis. The false-positiveand -negative rates of detection were 7.4 and 3.5%, respectively,for E. coli and 9.6 and 6.3%, respectively, for coliforms.

INTRODUCTION

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Introduction

In Taiwan, coliform bacteria are the recommended hygienic indicatororganisms for both raw and potable water. However, many bacteriain the genera of Klebsiella, Enterobacter, Citrobacter, andSerratia meet the definitional criteria for coliforms even thoughthey are not of fecal origin (8). Disagreements between governmentregulators and the water company sometimes relate to water thatcontains nonfecal coliforms, which interfere with the abilityto accurately detect coliforms of fecal origin (the permissiblelimit in Taiwan being 20,000 CFU per 100 ml of raw water). Assuggested previously, Escherichia coli could serve as a morespecific indicator for fecal contamination of freshwater. TheRepublic of China Environmental Protection Agency has approvedthe use of two methods for the detection and enumeration ofE. coli in water samples. One of them is a most-probable-numbermethod based on the defined substrate technology using o-nitrophenyl-ß-D-galactopyranoside(ONPG) and 4-methylumbelliferyl-ß-glucuronide (MUG)as the growth substrates (7).

A proprietary medium formulation and testing package incorporatingthese substrates has been marketed by IDEXX Laboratories (Westbrook,Maine) under the product name Colilert. The Colilert systemyields results that are consistently similar to those of traditionalstandard detection methods for E. coli and total coliforms infreshwater (4, 5, 6). Colilert-18, a system intended to providerapid (18-h) results, has been reported to detect coliformsand E. coli in potable water samples with an accuracy of 100%(10). However, other studies have disputed this degree of accuracy(14, 16). This dispute was of concern in our efforts to achieverapid and accurate E. coli and coliform detection in our watermonitoring program in Taiwan. Herein, we report the false-positiveand false-negative rates of E. coli and coliform detection insubtropical freshwater samples obtained near metropolitan Taipeiusing Colilert-18.

Isolation of target organisms.

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Isolation of target organisms.

Thirteen river water samples were collected from 11 differentsites between June 2002 and January 2003. At each sampling date,following at least 3 consecutive days of clear weather, watersamples were collected between 9 and 11 a.m. Water temperaturesin the region averaged 28.5 ± 1.8°C (July-September)and 22.3 ± 0.9°C (October-December). Each samplewas collected by immersing a 500-ml autoclaved polypropylenebottle in the middle of the river. The samples were placed onice and transported back to the laboratory. The entire process,from sampling to the commencement of incubation, took less than6 h. Detection of coliforms and E. coli by using the Colilert-18(Quanti-Tray/2000) method was done according to the manufacturer'sinstructions. Yellow wells indicated coliform bacteria, andwells that were yellow and fluorescent when exposed to UV light(366 nm) indicated E. coli. Quanti-Trays showing less than 20yellow wells (ONPG-positive) were selected for the recoveryof coliforms and E. coli.

A total of 640 wells, consisting of 224 yellow and fluorescentwells, 359 yellow wells, 54 colorless wells, and 3 colorlessand fluorescent wells, was selected for further study. Targetorganisms were recovered as described by Pisciotta et al. (14)by using sterile toothpicks to pierce the backing material ofeach Quanti-Tray. Liquid adhering to the toothpick was streakedonto m-ENDO (Difco, Detroit, Mich.) and m-TEC (Difco) agar platesfor coliform and E. coli isolation, respectively. The m-ENDOplates were incubated at 35°C for 24 h. The m-TEC agar plateswere incubated at 35 ± 0.5°C for 2 h and then transferredto 44.5 ± 0.2°C for 22 h. Organisms that formed yellowish(i.e., yellow, yellow-green, or yellow-brown) colonies on m-TECagar and showed negative urease activity were classified asE. coli.

Accuracy for E. coli detection.

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Accuracy for e. coli...

When m-TEC agar was used to isolate E. coli from the ONPG-positive,MUG-positive (yellow and fluorescent) wells, 40 wells failedto yield colonies with typical E. coli characteristics (Table1). Assuming that the false-negative rate for verified E. colion m-TEC agar is less than 1% (3), the false-positive rate was17.9% (40 out of 224) (Table 1).

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TABLE 1. Verification of presumptive E. coli and nontarget Colilert-18 wells

Dufour et al. (3) reported that for freshwater samples, only85% of the presumptive E. coli colonies that develop on m-TECagar can be verified as E. coli. Furthermore, the inabilityof E. coli to form yellow colonies on m-TEC agar may simplybe due to the absence of lactose permease (2). Fricker et al.(9) reported that approximately 10% of the coliforms isolatedfrom surface freshwater lack this enzyme. Alternatively, someE. coli may simply fail to express ß-galactosidaseactivity when incubated at 44.5°C (1). Therefore, furtherconfirmation is needed.

To verify the above results, presumed E. coli isolates wereisolated from 158 yellow and fluorescent wells by using m-TECagar and were identified on the basis of their fatty acid methylester (FAME) profiles (12). E. coli ATCC 11775 (American TypeCulture Collection, Manassas, Va.) was added to every tenthsample isolate to serve as a control. Of these presumed E. coliisolates, 56 could not be identified as E. coli. When these56 isolates were reinoculated into the Colilert-18 medium, 9isolates failed to develop E. coli characteristics. Hence, theywere excluded from further analysis. The remaining 47 isolateswere reidentified by using API 20E strips (bioMérieux,Inc., Hazelwood, Mo.) with E. coli ATCC 11775 as the control,and 36 isolates were positively identified as E. coli. Thus,in the present study, the use of Colilert-18 to survey subtropicalfreshwater produced a false-positive rate for E. coli detectionof 7.4% (Table 1). This rate is considerably lower than therate of 27.3% (3 out of 11 isolates) observed by Pisciotta etal. (14) when they used Colilert-18 and API 20E strips to assayfor E. coli in Florida freshwater samples.

Although ß-glucuronidase activity is used in the Colilert-18system as a benchmark for E. coli, a sizable proportion (10to 20%) of E. coli strains isolated from environmental sourcesare not MUG positive (17). Therefore, a false-negative evaluationis necessary. Suspensions from 416 wells, representing 359 yellowwells, 3 colorless-fluorescent wells, and 54 colorless wells,were plated onto m-TEC agar for isolation of E. coli. Of these416 samples, 46 urease-negative, yellowish colonies were obtained,representing a false-negative rate for E. coli of 11.1%. Strainidentification was also carried out to further confirm thisresult. Pure cultures were obtained from 283 Quanti-Tray wells,including 240 yellow wells, 3 colorless-fluorescent wells, and40 colorless wells. Twenty-one isolates were identified as E.coli on the basis of their FAME profiles. These isolates werechecked by growth in the Colilert-18 medium and subsequent API20E reidentification. Only 10 isolates (7 from yellow wellsand 3 from colorless-fluorescent wells) were actually E. coli.Therefore, the false-negative rate was approximately 3.5% (Table1). A markedly higher false-negative rate of 11% has been reportedfor Colilert-18 by Schets et al. (16). In their study, theyrelied chiefly on the properties of lactose utilization andindole production at 44 ± 0.5°C as confirmation ofE. coli. However, E. coli is not the only coliform with theability to produce indole at this temperature (18). The presentstudy's use of two direct identification systems may well producea more accurate assessment of Colilert-18 than has hithertobeen the case. If so, then the low false-negative rate shownin this study bodes well for the technique as a monitoring toolfor subtropical freshwater.

Accuracy for coliform detection.

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Accuracy for coliform detection.

For false-positive evaluation of coliforms, pure cultures wereisolated from 240 yellow wells and identified based on theirFAME profiles. Of the 23 isolates not identified as coliforms,16 were Erwinia carotovora. Hence, the false-positive rate forcoliform detection was about 9.6% (Table 2). On the other hand,of 32 isolates obtained from 350 colorless wells, 22 were coliforms,which makes the false-negative rate about 6.3%.

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TABLE 2. Verification of presumptive coliforms and nontarget Colilert-18 wells

Previous studies that used membrane filtration methods (m-FCand m-TEC) to enumerate fecal coliforms and indole-methyl red-Voges-Proskauer-citratetests and/or API 20E strips to confirm identification of fecalcoliforms (13, 15) reported false-positive rates of 13 to 16%in temperate water and 30 to 36% in tropical water. The samestudies reported false-negative rates of 1 to 2% in temperatewater and 18 to 21% in tropical water. The present rates aremarkedly lower than the rates for tropical water. Another study(11) using the m-ENDO method to detect total coliforms in surfacewater, with subsequent API 20E identification, reported a 29.0%false-positive rate. Our observed 9.7% false-positive rate stronglysupports our contention that the Colilert technology is a moreaccurate coliform detection system than membrane filtrationmethods.

It is clear that ß-galactosidase exists in many gram-positiveand gram-negative bacteria aside from E. coli (19). Hence, theColilert-18 system tends to give higher total coliform countsthan the traditional methods. In countries like Taiwan, wheretotal coliform number still serves as the sole bioindicatorfor both treated and untreated water, conclusions based on Colilertdata should be drawn with care. With this caveat, Colilert-18is a satisfactory method for rapid screening for fecal contaminationin subtropical freshwater.

ACKNOWLEDGMENTS

This study was supported by the Environmental Protection Agencyand the National Science Council, Republic of China (grantsEPA-90-U1G1-02-101 and NSC90-2745-P-031-004, respectively).

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, Soochow University, Shih Lin, Taiwan, Republic of China. Phone: 886-2-28812523. Fax: 886-2-28831193. E-mail: wlchao{at}mail.scu.edu.tw.

REFERENCES

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