Performance Validation of Chromogenic Coliform Agar For The Enumeration of Escherichia Coli [PDF]

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Letters in Applied Microbiology ISSN 0266-8254



ORIGINAL ARTICLE



Performance validation of chromogenic coliform agar for the enumeration of Escherichia coli and coliform bacteria B. Lange1, M. Strathmann2 and R. Oßmer3 1 Department of Water Quality, IWW Water Centre, Muelheim an der Ruhr, Germany 2 Department of Applied Microbiology, IWW Water Centre, Muelheim an der Ruhr, Germany 3 BioMonitoring R&D, Merck KGaA, Merck Millipore, Darmstadt, Germany



Significance and Impact of the Study: The international standard for the detection and enumeration of E. coli and coliform bacteria by membrane filtration (ISO 9308-1) is currently under revision and will be published in 2014. In the new standard, lactose–triphenyl tetrazolium chloride (TTC) agar will be replaced by a CCA. A performance validation of this revised method according to ENV ISO 13843 is presented in this study to determine fundamental data on its applicability and to provide reference data for secondary validation by users of this method.



Keywords chromogenic coliform agar, coliform bacteria, drinking water, Escherichia coli, performance, validation. Correspondence Bernd Lange, IWW Water Centre, Moritzstrasse 26, 45476 Muelheim an der Ruhr, Germany. E-mail: [email protected] 2013/1025: received 24 May 2013, revised 1 August 2013 and accepted 12 August 2013 doi:10.1111/lam.12147



Abstract The performance of chromogenic coliform agar (CCA) for the enumeration of Escherichia coli and coliform bacteria was validated according to ENV ISO 13843 using pure cultures and naturally contaminated water samples. The results indicate that for the detection of E. coli and coliform bacteria, respectively, the method is sensitive (94 and 91%), specific (97 and 94%), selective (selectivity 0
78 and 0
32) and efficient (96 and 92%). Relative recovery of E. coli and coliform bacteria on CCA in comparison with tryptone soy agar (TSA) was good (104 and 94% in mean, >80 and >70% in all cases), and repeatability and reproducibility were sufficient. The linear working range was defined for 10–100 total target colonies per 47-mm membrane filter. A high precision of the method was confirmed by low overdispersion in comparison with Poisson distribution. The robustness of the method with respect to the variable incubation time of 21  3 h was found to be low, because an incidental increase in presumptive colonies especially between 18 and 21 h was observed. In conclusion, the CCA method was proved as a reliable method for the quantification of E. coli and coliform bacteria.



Introduction The presence and extent of faecal contamination is an important issue for the assessment of the quality of a water sample and of the risk to human health due to infection. It is widely accepted in practice and in literature to use Escherichia coli and coliform bacteria as indicators for a faecal pollution (Byamukama et al. 2000; Geissler et al. 2000). Other coliform bacteria than E. coli can also be found in the environment, for example soil or surface water (Geissler et al. 2000). This makes interpretation of results for coliform bacteria more difficult, but the



presence of coliform bacteria, although not a proof of faecal contamination, may indicate problems in the treatment process or during drinking water distribution. To enable a reliable detection and quantification of E. coli and coliform bacteria, an appropriate analytical method is necessary. Different methods were reviewed in the study by Rompr
e et al. (2002). ISO/DIS 9308-1 (2012) specifies a method for the enumeration of E. coli and coliform bacteria based on membrane filtration and subsequent culture on a chromogenic agar medium. It is especially suitable for water samples with low bacterial numbers (e.g. drinking water,



Letters in Applied Microbiology 57, 547--553 © 2013 The Society for Applied Microbiology



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disinfected pool water or finished water from drinking water treatment plants). Due to the low selectivity of the differential agar medium, background growth can interfere with the reliable enumeration of coliform bacteria and E. coli, for example in surface waters or shallow well waters. This method is not suitable for these types of water. The current third version of ISO 9308-1 cancels and replaces the second edition (ISO 9308-1 2000), which has been technically revised. This revision was necessary, because the previous version was based on the use of lactose–TTC agar and showed too low selectivity and problems with high accompanying flora (Pitk€anen et al. 2007). In that method, the detection of coliform bacteria relies on lactose fermentation and negative oxidase reaction and of E. coli on the detection of tryptophanase by indole reaction. In addition, on ISO level, it was decided already in 2006 to define coliform bacteria on their b-D-galactosidase activity (EC 3.2.1.23) and no longer on lactose fermentation. Further, E. coli should be identified by its additional b-D-glucuronidase activity (EC 3.2.1.31). The membrane filtration method provided in ISO/DIS 9308-1 (2012) meets these requirements. This method is based on the following principles: (i) the use of a suitable growth medium containing peptones, pyruvate, sorbitol and phosphate buffer to support rapid colony growth, even for the sublethally injured coliforms, (ii) inhibition of accompanying Gram-positive bacteria flora as well as some Gram-negative bacteria flora by the use of Tergitol® 7, which has no negative effect on the growth of the coliform bacteria, (iii) the use of Salmon-GAL (6-chloro3-indoxyl-ß-D-galactopyranoside) and isopropyl-b-D-thiogalactopyranoside (IPTG) substrate, which can be cleaved by b-D-galactosidase, resulting in a pink to red coloration of the coliform colonies and (iv) the substrate X-glucuronide (5-bromo-4-chloro-3-indoxyl-ß-D-glucuronide) for the detection of b-D-glucuronidase. Escherichia coli cleaves both Salmon-GAL and X-glucuronide; thus, positive colonies will show a dark blue to violet colour. The performance of the ISO/DIS 9308-1 (2012) method was validated in this study according to the requirements of ENV ISO 13843 using selected pure cultures and water samples contaminated with ambient river water. Results and discussion Target organism identification Pure culture challenge Typical characteristic reactions of target and nontarget bacteria were confirmed using bacterial suspensions of pure cultures of selected reference strains of E. coli, coliform bacteria and noncoliform Gram-negative bacteria 548



for membrane filtration and cultivation on CCA. Typical results are shown in Fig. 1. In the case of E. coli, all tested strains revealed dark-blue- to violet-coloured colonies, and in the case of coliform bacteria, pink- to red-coloured colonies. Nontarget bacteria produced beige- to yellow-coloured colonies or even no growth on the CCA medium. In conclusion, using the selected chromogenic enzyme substrates contained in the CCA medium, a clear identification and differentiation of the two groups of target organisms and nontarget flora were possible. Sensitivity, specificity and selectivity studies The fundamental parameters of the method were determined as described in ENV ISO 13843 section 9.2. Drinking water spiked with naturally contaminated ambient river water samples from 10 different locations, each having different levels of contamination, was used for these studies, covering concentrations within the whole working range of the method. Fifteen different independent samples were processed according to ISO/DIS 9308-1 (2012), and in total, 220 colonies were randomly selected, including typical E. coli and coliform bacteria and atypical colonies. All colonies were tested regarding their oxidase activity, Gram staining, cell morphology, b-D-galactosidase and b-D-glucuronidase activity. Using these results, it was possible to group the colonies into four groups: (i) true positives, (ii) false negatives, (iii) false positives and (iv) true negatives. The results obtained for readings of coliform bacteria (incl. E. coli) and E. coli are compiled in Tables 1 and 2, respectively, and were used to calculate sensitivity, specificity, false-positive rate, false-negative rate, efficacy and selectivity (Table 3). The raw data of all tested colonies are compiled in the supporting information Table S1. Six and four colonies from the 117 and 65 typical colonies for coliform bacteria and E. coli, respectively, were identified as noncoliform species. Eleven and four colonies from the 103 and 155 atypical colonies were actually found to be coliform bacteria and E. coli, respectively. In general, performance results were better for the detection of E. coli compared with total coliform bacteria. The outcomes of this validation indicate that the ISO/DIS 9308-1 (2012) method is sensitive (94 and 91%) and specific (97 and 94%) for the detection of E. coli and coliform bacteria, respectively. The method is also selective, having a value of 0
32 and 0
78 for coliform bacteria and E. coli, respectively, which is in both cases considerably better than the guidance value of 1 suggested by ENV ISO 13843 for colony count methods (data not shown). It has to be noted that selectivity for E. coli seems to be worse in comparison with coliform bacteria. This is due to the fact that the CCA medium is not intended to be selective for E. coli alone, but for total



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Performance of chromogenic coliform agar



(a)



(b)



(c)



(d)



(e)



(f)



Figure 1 Typical appearance of colonies of target and nontarget organisms grown on membrane filters on chromogenic coliform agar (CCA); (a) Escherichia coli WDCM 00012 (target organism), (b) Citrobacter freundii DSM 30039 (target organism for coliform bacteria), (c) Enterobacter aerogenes WDCM 00175 (target organism for coliform bacteria); (d) Aeromonas hydrophila WDCM 00063 (nontarget organism), (e) Staphylococcus aureus WDCM 00032 (nontarget organism); (f) Naturally contaminated water sample obtained from the River Ruhr.



Table 1 Experimental data for the determination of coliform bacteria obtained for randomly selected typical and atypical colonies (n = 220)



Table 2 Experimental data for the determination of E. coli obtained for randomly selected typical and atypical colonies (n = 220)



Presumptive colonies



Presumptive colonies



Confirmed colonies Positive Negative Total



Typical



Atypical



Total



111 6 117



11 92 103



122 98 220



Confirmed colonies Positive Negative Total



Letters in Applied Microbiology 57, 547--553 © 2013 The Society for Applied Microbiology



Typical



Atypical



Total



61 4 65



4 151 155



65 155 220



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Table 3 Performance characteristics for the use of chromogenic coliform agar for the enumeration of Escherichia coli and coliform bacteria according to ISO/DIS 9308-1 (2012)



Parameter Identification* Sensitivity Specificity False-positive rate False-negative rate Efficiency Selectivity Counting uncertainty (RSD) Repeatability Reproducibility Recovery Range of quantitative determination (colonies per 47-mm membrane filter) Robustness of incubation time



E. coli



Coliform bacteria



93
8% 97
4% 6
2% 2
6% 96
4% 0
78



91
0% 93
9% 5
1% 10
7% 92
3% 0
32



0
046 0
127 >80% 10–100



0
035 0
114 >70% 10–100



Incidental increase in presumptive colonies especially between 18 and 21 h



RSD, relative standard deviations. *Number of investigated colonies: n = 220.



coliform bacteria including E. coli. With a calculated efficiency value of 96 and 92% for the detection of E. coli and coliform bacteria, respectively, the data suggest that the method is highly efficient. False-negative results as well as false-positive results were considerably low. Relative recovery The recovery of target organisms grown on CCA was determined relatively in comparison with the same samples cultivated on nonselective TSA medium. Two E. coli strains and four coliform bacteria that are not E. coli were tested, each in at least ten replicate sample filtrations. Mean relative recovery rates on CCA of 104 and 94% for E. coli and coliform bacteria (incl. E. coli) were observed, indicating a high recovery of target organisms in general. In all cases, relative recovery was >70% for coliform bacteria and >80% for E. coli (Table 3). Raw data for determination of the relative recovery rates are summarized in the supporting information Table S2. Counting uncertainty Repeatability is the agreement in counts obtained by repeated counting performed by the same analyst, and reproducibility is the agreement between subsequent counts obtained by two or more different analysts. ENV ISO 13843 Annex B provides guidance on the assessment 550



of counting repeatability and reproducibility using the relative standard deviations (RSD) of repeated counts and recommends that when using mixed populations, RSD should ideally be 95%, whereas the counting of coliform bacteria colonies showed lower linearity of >50% in this range (data not shown). In conclusion, 10–100 total target colonies per 47-mm membrane filter



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Performance of chromogenic coliform agar



400 350



y = 2·6222x R ² = 0·9856



Colony counts



300



Figure 2 Linear working range of the method using drinking water spiked with a naturally contaminated surface water sample obtained from the River Ruhr; mean values of three independent counts per sample are shown: (♦) Escherichia coli, (■) coliform bacteria, ( ) total colony counts.



250 y = 2·2715x R ² = 0·9991



200 150 100



y = 0·7572x R ² = 0·9965



50 0 0



16



32



△



can be defined as a suitable working range for the CCA method according to ISO/DIS 9308-1 (2012). Because E. coli and other coliform bacteria are determined on the same filter, both will contribute to this number of colonies. In dependence on the ratio of E. coli to other coliform bacteria, the countable number of E. coli will vary with respect to the other coliform colonies. Precision In general, precision is defined as the closeness of agreement between independent test results obtained under stipulated conditions. For microbial assays, a basic random variation is unavoidable due to the random distribution of bacteria in the tested sample as described by the Poisson distribution. Technical imperfections (e.g. variation in media and sample processing) and other causes (e.g. variations in laboratory staff) are responsible for additional variation. Parallel determinations can vary even more than is explained by the Poisson distribution. This situation is called overdispersion. Parallel determinations involving the whole analytical procedure cannot be expected to follow the Poisson distribution. The quantification of the overdispersion can be used as a means to describe the precision of an analytical procedure. In this study, the overdispersion at detector level was determined from 10 drinking water samples spiked with naturally contaminated river water samples, each processed tenfold in parallel, and each counted by at least two different technicians. Quantitative interpretation of the overdispersion was performed by (i) calculation of the Poisson index of dispersion (Χ²n 1), (ii) calculation of the overdispersion coefficient (u) by Anscombe’s method I (Anscombe 1950) and (iii) using the regression approach described in ENV ISO 13843 section 6.2.3.



48



64



80



96



112



128



Relative sample volume



Detailed data on these calculations can be found in the supporting information Table S6. In summary, for the determination of E. coli and coliform bacteria, overdispersion was found to be 9
3 and 3
1% in excess of the Poisson distribution, respectively, in mean of the ten investigated samples. Using the regression approach to take into account the whole set of investigated samples, an overdispersion of 16
6 and 8
4% was observed for the determination of E. coli and coliform bacteria, respectively. These values indicate only minor deviation of uncertainty of results obtained by CCA method from the theoretically expected one due to Poisson distribution, thus proving a good precision of the method. Robustness of incubation time Robustness means tolerance towards slight changes in procedure or towards unavoidable variations in conditions of the laboratory environment. The aspect of robustness that is of major relevance for the CCA method is the variance of incubation period of 21  3 h, as described in ISO/DIS 9308-1 (2012). To investigate this influence, six samples of drinking water spiked with different naturally contaminated surface water samples were processed in tenfold replicates and were each counted after 18, 21 and 24 h (raw data and significance values are presented in the supporting information Table S5). In the case of determination of E. coli, an increase in presumptive colonies of 11
0% (6
6%) was observed between 18 and 21 h, whereas the increase between 21 and 24 h was comparably low [1
4% (3
1%)]. For quantification of coliform bacteria (incl. E. coli), an increase of 28
6% (10
9%) and 18
4% (8
3%) was determined between 18-h and 21-h incubation time, and 21 h and 24 h incubation time, respectively.



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The data suggest that an incidental increase in presumptive colonies especially between 18 and 21 h will occur for both E. coli and other coliform bacteria. For E. coli, no significant change in presumptive colonies was observed between 21 and 24 h, and thus, the incubation time is robust in this range. However, for some coliform bacteria that either grow slowly or have reduced b-Dgalactosidase activity, there may be a significant increase in counts within the accepted incubation time. An increase in counts over a prescribed incubation period is a well-recognized phenomenon (e.g. for typical membrane filtration methods, such as ISO 7899-2 for enterococci, unpublished data). However, provided that the increase in counts after an extended incubation is not excessive, for practical and operational considerations, counts from the CCA method at 21  3 h of incubation can be considered acceptable. Nevertheless, the preferred incubation time for water samples should be 21 h. Incubation for 24 h increases the recovery of the target bacteria especially if they are stressed, for example, from disinfected waters. Materials and methods Bacterial strains The following bacterial strains were used in this study: E. coli WDCM 00012, Citrobacter freundii DSM 30039, Enterobacter aerogenes WDCM 00175, Aeromonas hydrophila WDCM 00063 and Pseudomonas aeruginosa WDCM 00024 were obtained from the German Collection of Micro-organisms and Cell Cultures (DSMZ, Braunschweig, Germany) and E. coli and Klebsiella pneumoniae round robin strains from the Nieders€achsisches Landesgesundheitsamt (NLGA, Aurich, Germany). All microorganisms were maintained on TSA.



samples were pretested by Colilertâ-18 QuantiTray 2000 (IDEXX, Ludwigsburg, Germany) according to the manufacturer’s instructions, and dilutions were adapted to obtain required target colony counts. Detection of Escherichia coli and coliform bacteria The quantification of E. coli and coliform bacteria was performed using the ISO/DIS 9308-1 (2012) method. Briefly, samples (10–100 ml) were vacuum-filtered through a membrane composed of cellulose mixed ester (0
45 lm pore size, EZPak, Merck, cat. no. EZHAWG474), placed on CCA plates (Chromocult® coliform agar, Merck) and incubated for (21  3) h at (36  2)°C. For some experiments (determination of robustness), cultivation time was varied to exactly 18, 21 and 24 h. For some experiments (determination of relative recovery), cultivation was performed on nonselective TSA medium. Counting of b-D-galactosidase-positive colonies (pink to red) resulted in presumptive coliform bacteria that are not E. coli. To avoid false-positive results, caused by oxidase-positive bacteria such as Aeromonas spp, the presumptive colonies were confirmed by a negative oxidase reaction. Escherichia coli was quantified by counting ßgalactosidase- and ß-glucuronidase-positive colonies (dark blue to violet). Total coliform bacteria were calculated as the sum of oxidase-negative colonies with pink to red colour and all dark blue to violet colonies. Colonies having other colours than typical for E. coli or coliform bacteria were counted as ‘atypical colonies’. Oxidase test The oxidase activity of presumptive colonies was tested using Bactident® Oxidase test (Merck, cat. no. 1.13300.0001) according to the manufacturer’s instructions.



Culture media Chromogenic coliform agar (CCA) was obtained from Merck (Chromocult® coliform agar, cat. no. 1.10426.0500, Merck Millipore, Germany). TSA was obtained from Oxoid (cat. no. CM0131). All culture media were prepared in accordance with manufacturer’s instructions.



Gram staining Gram staining was performed using Gram-color gram staining kit (Merck, cat. no. 1.11885.0001) according to the manufacturer’s instructions. Samples were investigated using a Leica Laborlux microscope at 1000-fold magnification.



Water samples All water samples investigated throughout this study were prepared using drinking water (tap water, M€ ulheim an der Ruhr, Germany) spiked with naturally contaminated ambient river water samples derived from 10 different sampling sites at surface waters located in M€ ulheim an der Ruhr (Germany) and Duisburg (Germany). Concentrations of E. coli and coliform bacteria in river water 552



Detection of b-D-galactosidase and b-D-glucuronidase activity The principle of the method described in ISO 9308-2 (2012) was used to detect the presence or absence o b-D-galactosidase and b-D-glucuronidase activity of presumptive colonies. The proprietary Colilertâ-18 substrate (IDEXX, cat. no. WP200I) was dissolved in 100 ml sterile



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water and was subsequently divided into portions of 5 ml in test tubes. These test tubes were inoculated with material obtained from the presumptive colonies, mixed and incubated for 20  2 h at 36  1°C. Detection was recorded as presence/absence of yellow colour for b-Dgalactosidase activity and blue fluorescence (excitation at 366 nm) for b-D-glucuronidase activity. Identification of isolates Some presumptive coliform colonies (red) that were tested oxidase positive were identified using the proprietary APIâ 20 E system (Biom
erieux SA, Marcy l’Etoile, France) according to the manufacturer’s instructions. Data analysis All data analyses were performed using Microsoft Excel (version 2010). Acknowledgement We thank Fabian Ansorge for technical assistance. Conflict of Interest Dr. Oßmer is an employee of the Merck KGaA. Mr. Lange and Dr. Strathmann have no conflict of interest. References Anscombe, F.J. (1950) Sampling theory of the negative binomial and logarithmic series distributions. Biometrika 37, 358–382. Byamukama, D., Kansiime, F., Mach, R.L. and Farnleitner, A.H. (2000) Determination of Escherichia coli contamination with Chromocult Coliform Agar showed a high level of discrimination efficiency for differing fecal pollution levels in tropical waters of Kampala, Uganda. Appl Environ Microbiol 66, 864–868. ENV ISO 13843 (2001) Water Quality - Guidance on Validation of Microbiological Methods. Geneva: International Standards Organization.



Performance of chromogenic coliform agar



Geissler, K., Manafi, M., Amoros, I. and Alonso, J.L. (2000) Quantitative determination of total coliforms and Escherichia coli in marine waters with chromogenic and fluorogenic media. J Appl Microbiol 88, 280–285. ISO 9308-1 (2000) Water Quality - Enumeration of Escherichia coli and Coliform Bacteria - Part 1: Membrane Filtration Method. Geneva: International Standards Organization. ISO 9308-2 (2012) Water Quality - Enumeration of Escherichia coli and Coliform Bacteria - Part 2: Most Probable Number Method. Geneva: International Standards Organization. ISO/DIS 9308-1 (2012) Water Quality - Enumeration of Escherichia coli and Coliform Bacteria - Part 1: Membrane Filtration Method for Waters with Low Bacterial Background Flora. Geneva: International Standards Organization. Pitk€anen, T., Paakkari, P., Miettinen, I.T., Heinonen-Tanski, H., Paulin, L. and H€anninen, M.-L. (2007) Comparison of media for enumeration of coliform bacteria and Escherichia coli in non-disinfected water. J Microbiol Methods 68, 522–529. Rompr
e, A., Servais, P., Baudart, J., de-Roubin, M.R. and Laurent, P. (2002) Detection and enumeration of coliforms in drinking water: current methods and emerging approaches. J Microbiol Methods 49, 31–54.



Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1 Specificity and selectivity for the determination of E. coli and coliform bacteria using CCA. Table S2 Recovery rates for the determination of E. coli and coliform bacteria using CCA. Table S3 Repeatability for the determination of E. coli and coliform bacteria using CCA. Table S4 Reproducibility for the determination of E. coli and coliform bacteria using CCA. Table S5 Robustness of incubation time for the determination of E. coli and coliform bacteria using CCA. Table S6 Precision testing for the determination of E. coli and coliform bacteria using CCA.



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