International Journal of Food Microbiology 68 2001. 217223 www.elsevier.

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An alternative approach for enumeration of Escherichia coli in foods

Ibrahim Cakir a , Hilal B. Dogan a , A. Kadir Halkman a , Randy W. Worobo b,)
b a Department of Food Engineering, Faculty of Agriculture, Ankara Uniersity, Diskapi 06110, Ankara, Turkey Department of Food Science and Technology, Cornell Uniersity, New York State Agricultural Experiment Station (NYSAES), Genea, NY 14456-0462, USA

Received 4 August 2000; received in revised form 14 December 2000; accepted 5 February 2001

Abstract An assay to screen Escherichia coli in foods using MUG supplemented lauryl sulfate tryptose LST. broth instead of tryptone water TW. medium was evaluated. The results presented in this paper suggest that this method is more sensitive for lower levels of E. coli, faster 1618 h vs. 610 days. and less expensive 2.454 vs. 2.887 EUROrsample. than the standard ISO procedure. Thus, this method may be beneficial for use when both fecal coliforms and E. coli analyses are required in food systems. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: E. coli; Fecal coliforms; MUG; Analysis

1. Introduction Samples containing coliforms but free of Escherichia coli comprise the majority of samples encountered in a food microbiology laboratory. Positive coliform food samples may be due to non-fecal Enterobacteriaceae such as Enterobacter aerogenes, Enterobacter cloacae and Klebsiella pneumoniae. Therefore, the analysis for E. coli provides absolute proof of fecal contamination Hitchins et al., 1992.. The analysis time for the determination of E. coli is 6 days by standard International Standard Organization ISO. methods and 10 days by standard Associa) Corresponding author. Tel.: q 1-315-787-2279; fax: q 1-315787-2284. E-mail address: rww8@cornell.edu R.W. Worobo..

tion of Official Analytical Chemist AOAC. methods. Generally, the modified most probable number methods based on the incorporation of 4-methylumbelliferyl-b-D-glucuronic acid MUG. enumerates only E. coli as an indicator of fecal contamination. This technique is acceptable for the food industry but in some instances, it is required to analyze for both fecal coliforms and E. coli. An assay to determine the presence of fecal coliforms and E. coli can be used to assess the sanitary quality of foods. The most probable number MPN. technique which is recommended by the AOAC and ISO for the enumeration of fecal coliforms, takes 4 days while the confirmatory tests for E. coli requires an additional 6 days by AOAC methods and 2 days by ISO method Poelma et al., 1987; Anonymous, 1993; Jay, 1996..

0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 0 1 . 0 0 4 8 8 - 3

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Since rapid methods for E. coli enumeration are needed by the food industry, efforts have been made to develop them. Amongst them, MUG technique was evaluated as a rapid, specific, inexpensive and sensitive method Robison, 1984.. MUG can be used with both liquid and solid media as well as for membrane-filter methods. Some rapid presenceabsence test kits and rapid identification kits based on MUG substrate are also commercially available Moberg, 1985; Entis and Boleszczuk, 1990; Feng, 1998.. There is a close relationship between fecal coliforms and E. coli. However, with analyses and the interpretation of analyses of coliforms, caution must be taken. With the fecal coliform test it is possible that non-fecal coliforms are capable of growing and leading to false positive reactions for non-fecal coliforms. Fruits and vegetables commonly contain non-fecal coliforms as part of their normal microflora but it is not an indication of fecal contamination Splittstoesser et al., 1980.. In situations such as these, standard AOAC methods for E. coli analysis using MUG supplemented media and observing fluorescence is a better indicator for fecal contamination because this method enumerates only for E. coli and eliminates the possibility for non-fecal coliform false positives Hitchins et al., 1992.. Fecal coliform analysis is a useful analysis for shellfish growing waters and in some national and international trade, where buyers or importers demand additional fecal coliform analysis Peterson et al., 1987; Matner et al., 1990; Anonymous, 1993; Harrigan, 1998.. This study is part of a larger project entitled AResearch on Fecal Coliforms in FoodsB. For the present project, a total of 500 food samples from 10 different kinds of foods were analyzed by standard ISO method vs. MUG technique for the presence of contamination with fecal coliforms and E. coli. These samples also were analyzed for both fecal coliforms and E. coli to reduce total analysis time from 6 to 5 days using an economical approach. 2. Materials and methods 2.1. Food sampling for coliform analyses Five hundred food samples consisting of pasteurized milk, yogurt, butter, cheese, ice cream, salad, delicatessen products, cream sweet. cake, spices and

fresh fruits and vegetables 50 random different samples of each food. were analyzed for the presence of E. coli. All the samples were collected from different Turkish retail grocery stores located within Ankara. The various samples were immediately refrigerated and subjected for analysis within 4 h. 2.2. Fecal coliform and E. coli analyses A schematic diagram used to determine the efficiency of MUG method is shown in Fig. 1. All samples 10 g or ml. were homogenized by blending and then 10-fold serially diluted in 0.85% NaCl solution. Five consecutive dilutions from 10 0 to 10y4 for liquid samples and from 10y1 to 10y5 for solid samples. of 1 ml were used to inoculate three tubes containing lauryl sulfate tryptose LST. broth Merck, Ankara, Turkey. and incubated at 378C for 48 h. Three out of five consecutive dilutions were used for MPN enumeration according to

Fig. 1. Flow diagram for testing the presence of E. coli using the MUG method.

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ISO methods. All the gas positive tubes were then subcultured using a 5-mm inoculating loop in EC broth Merck. and incubated in a water bath at 44.58C for 48 h. After the incubation period, the tubes were observed for gas production. The gas positive tubes were then subcultured using a loop full of a 5-mm inoculating loop into 10 ml of tryptone water TW. Merck. and incubated at 44.58C in a water bath for 48 h. As a confirmation test for the presence of E. coli, an indole test was performed by taking the culture media and adding 0.2 ml of dimethylaminobenzaldehyde and looking for the development of red color that is indicative of a positive indole reaction. Alternatively, the same EC broth positive tubes were subcultured using a loop full of a 5-mm inoculating loop into 2.5 ml LST broth q MUG medium Merck. and incubated at 378C for 24 h. These tubes were examined for blue fluorescence at a wavelength of 366 nm using a hand held UV lamp Merck.. Slightly fluorescent tubes were retested to clarify their fluorescence reaction. The glass tubes used for observing fluorescence were prechecked to ensure no fluorescence was emitted due to the glass itself. To determine non-E. coli coliforms in the food samples, EC broth positive tubes were surface plated on violet red bile agar VRBA. supplemented with MUG Merck.. After incubation at 378C for 24 h, fluorescent negative colonies were picked and identified using indole, methyl red, VoguesProskauer and citrate reactions followed by carbohydrate fermentation test. Using this procedure, Clark and El-Shaarawi 1993. determined that the fluorescence response appeared more quickly than the gas response. 2.3. Statistical analyses Log 10 values of MUG and ISO results were statistically compared by Pearsons correlation coefficient r ., Cronbachs alpha a . and determination coefficient r 2 . analyses. The statistical package program SPSS 9.0 SPSS, Chicago, IL. for windows was used. The reliability analyses are explained as follows: Pearsons correlation coefficient r .: measures the degree of linearity between two variables such as x and y actually MUG and ISO results..

Cronbachs alpha a .: it is the repeatability of the measure between two groups or variables. If all items are perfectly reliable and measure the same variable, then Cronbachs alpha coefficient will be equal to 1.000. Determination coefficient r 2 .: determines the variation in one of the variables by the variation of the other variable. This is square of Pearsons correlation coefficient Winer, 1971..

3. Results 3.1. Fecal coliform and E. coli detection Of the 1083 food samples analyzed, uncountable high, low values and suspect results were excluded, yielding the results of 500 food sample analyses. Fecal coliforms were identified in all 500 food samples with a range of 0.185.04 log MPNrg or ml. However, E. coli was only identified in 467 93.4%. food samples ranging from 0.185.04 log MPNrg or ml. 3.2. Statistical analyses of the different enumeration methods The results of the reliability analysis of ISO vs. 2.5-ml MUG method are given in Table 1. AccordTable 1 Reliability analyses for the ISO vs. alternative 2.5-ml MUG method counts by log 10 values. Food type Pearsons correlation r. 0.9932 0.9903 0.9957 0.9918 0.9988 0.9794 0.9972 0.9894 0.9811 0.9975 0.9924 Determination coefficient r2. 0.9864 0.9807 0.9914 0.9837 0.9976 0.9592 0.9944 0.9789 0.9626 0.9950 0.9849 Cronbachs alpha a . 0.9966 0.9951 0.9979 0.9959 0.9994 0.9896 0.9986 0.9947 0.9904 0.9988 0.9962

Pasteurized milk Yoghurt Cheese Butter Ice cream Salad Delicatessen products Cream cake Spices Fruitvegetable TOTAL

For all the figures, p- 0.0001.

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I. Cakir et al.r International Journal of Food Microbiology 68 (2001) 217223 Table 2 continued . Standard ISO method 4.04 4.38 4.66 5.04 Total Alternative method 4.04 4.38 4.66 5.04 Number of samples 13 4 4 9 500 Difference between methods 0 0 0 0

Table 2 Log MPN results and differences of ISO and alternative 2.5-ml MUG methods Standard ISO method 0 0 0 0 0 0.18 0.32 0.36 0.56 0.56 0.56 0.56 0.56 0.63 0.87 0.87 0.96 0.96 0.97 0.97 0.97 1.18 1.18 1.32 1.36 1.36 1.36 1.36 1.38 1.63 1.63 1.63 1.66 1.97 1.97 1.97 2.04 2.04 2.18 2.38 2.38 2.38 2.66 3.04 3.04 3.04 3.38 3.38 3.66 3.66 Alternative method 0 0.18 0.56 0.96 1.18 0.18 0.32 0.36 0.56 0.87 0.96 1.18 1.36 0.63 0.87 0.96 0.96 1.18 0.97 1.18 1.38 1.18 1.97 1.32 1.36 1.63 1.97 2.18 1.38 1.36 1.63 1.97 1.66 1.63 1.97 2.38 2.04 2.38 2.18 2.18 2.38 2.66 2.66 2.66 3.04 3.38 3.38 3.66 3.66 4.04 Number of samples 0 1 4 1 1 7 8 18 28 1 2 1 1 15 17 2 35 1 5 1 2 6 2 3 61 7 1 1 7 4 32 5 3 1 43 4 1 1 8 1 32 1 30 1 30 3 16 1 13 1 Difference between methods 0.18 0.56 0.96 0.18 0 0 0 0 0.31 0.40 0.62 0.80 0 0 0.09 0 0.22 0 0.21 0.41 0 0.79 0 0 0.27 0.61 0.82 0 0.27 0 0.34 0 0.34 0 0.41 0 0.34 0 0.20 0 0.28 0 0.38 0 0.34 0 0.28 0 0.38

ing to all three reliability tests, there is no statistical difference between the ISO method and the 2.5-ml MUG method. Table 2 illustrates the differences between the two methods. Here, 448 out of 500 samples 89.6%. gave the same results and the remaining 52 10.4%. were different. Only seven results 1.4% of total. gave higher counts by the ISO method while 45 higher results 9.0% of total. were obtained by the 2.5-ml MUG method. In other words, although statistical reliability tests show clearly that there are no significant statistical differences between the methods, the 2.5-ml MUG method appeared to be more sensitive 6.4 times. than the ISO method. Additionally, there were no negative results 0 log MPNrg or ml. by

Table 3 Cost analysis of different methods for the determination of fecal coliforms and E. coli Method (A) Standard ISO LST Broth EC Broth TW Medium Indole Reagent ) TOTAL (B) Modified MPN LST BrothqMUG EC Broth TOTAL (C) Alternatie MUG LST Broth EC Broth 2.5 ml LST BrothqMUG TOTAL Price EUROs. 0.819 1.141 0.472 4.826 0.455. 7.258 2.887.

2.223 1.141 3.363

0.819 1.141 0.494 2.454

) Indole reagent used as a ready to use left. or prepared by formulation right..

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the 2.5-ml MUG method while the ISO method had seven negative results ) 0 log MPNrg or ml.. It is important to point out that all the conflicting results were obtained in samples having lower counts of E. coli. 3.3. Cost analyses for E. coli and fecal coliform determination The total cost for each method is illustrated in Table 3. The range in price for the indole reagent represents the cost of commercially supplied first, followed by the laboratory prepared solution. The cost for each of the various media and solutions are calculated in EURO dollars. Where it is not specifically stated, 10 ml of media were used for the cost determination.

4. Discussion There have been numerous studies using the MUG method. Most of the research has found that the MUG technique is equal to or better than the traditional MPN method Robison, 1984; Moberg, 1985; Singh and Ng, 1986; Poelma et al., 1987; Moberg et al., 1988. and membrane filter method Entis and Boleszczuk, 1990; Cengi et al., 1993.. Additionally, the MUG method was evaluated to be a faster, less expensive, accurate, and simpler to perform. The only food sample that the MUG method is not suitable for is oysters. Oysters contain endogenous glucuronidase that interferes with the MUG test results. Modification of the assay by incorporating MUG into EC broth rather than LST broth eliminates this interference Koburger and Miller, 1985.. Autofluorescence of the glass tubes, negative bglucuronidase b-GUR. reaction of some enteropathogenic strains of E. coli and difficulty in reading the fluorogenic reaction are disadvantages of the MUG method, but due to the major advantages of this method, it is a reliable alternative to the current AOAC method for the enumeration of E. coli in foods Poelma et al., 1987.. As mentioned above, it has clearly been shown by previous research that after direct use of LST broth q MUG or after determining the E. coli count by the MUG reaction, subculturing the gas positive

tubes to EC broth is adequate for enumeration of both E. coli and fecal coliforms in 4 days by the modified MPN technique. In this manner, contrary to the standard MPN method, E. coli is enumerated before fecal coliforms and provides proof of fecal contamination with a shorter analysis time. The comparison of standard methods and the alternative 2.5-ml LST q MUG method showed no statistical difference between the methods for the detection of E. coli in foods. In other words, these methods can be used alternatively to each other Winer, 1971.. Since all the reliability tests showed no significant statistical difference p - 0.0001. between ISO and 2.5-ml MUG method for enumeration of E. coli in all food groups tested, there is no need to discuss the differences between the reliability results according to the different food groups. The lowest value r 2 s 0.9592. was for salads which also determined a high correlation between these counts. While values of 0.75 and higher for reliability tests indicate a high correlation for biological measurements, these very high values in our results ) 0.9. may be explained by the general characteristics of MPN technique and due to the use of log 10 values for the statistical analyses. As depicted in Table 3, the cost of analysis differs for each of the methods. The highest cost was for the standard ISO method which was 7.258 EUROranalysis but if laboratory prepared indole reagent was used, then the total cost decreased to 2.887 EUROranalysis. Even then, the 2.5-ml MUG method is less expensive than both the standard ISO method and the modified MPN method. The cost of the 2.5-ml MUG method is 85.0% of the standard ISO method considering that indole reagent was prepared in laboratory. and 73.0% of the modified MPN method. In addition, there is also an energy savings with the 2.5-ml MUG method since the incubations are carried out at 378C for 24 h vs. 44.58C for 48 h following the recommended procedures for the ISO method. These parameters are important factors for the food industry. A savings of 15.0% in the analysis cost plus energy saving. and 16.7% for the analysis time. These savings are also beneficial for official analyses by governmental and private analysis laboratories. This economical analysis is based on the enumeration of both fecal coliforms and E. coli. However,

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existing literature and this research suggest that only fecal coliforms or E. coli analysis is adequate. The modified MUG technique for determining only E. coli requires only 1 day of analysis time, 2.223 EUROrsample. and an incubator at 378C. The above comparison is based on the cases for which both fecal coliforms and E. coli analysis are required. These advantages are not comparable when only fecal coliforms or E. coli analyses is required. Four hundred and sixty seven 93.4%. samples yielded fluorescent positive colonies indicating that there were E. coli in all these samples at different levels. The MUG method was capable of detecting all of them but the ISO method was not successful in 22 4.4%. of the samples. These results show that the MUG-5 day method gave better results than the 6-day ISO method. The cost comparison of the standard ISO method, 2.5-ml MUG method and modified MPN method were compared Table 3., keeping in mind that the results for the three methods are approximately the same. For the formulation of Table 3, it was considered that: a. 10 ml of other broths was used while in the alternative method only 2.5 ml LST broth q MUG media was used; b. 1 ml of indole reagent was used for indole test. Indole reagent may be used in ready to use form 100-ml bottle. or may be prepared in the laboratory according to the formula; the latter is significantly cheaper than the former; c. nine LST broth tubes were inoculated at the first stage, eight of them subsequently inoculated to EC broth and all the EC broth tubes were analyzed for E. coli; d. The retail market prices in EURO. of Merck media in Ankara, Turkey were used for this cost determination. It is clear that the analysis of both fecal coliforms and E. coli in the same food sample seems not worthy of recommendation, but in the food industry, it is sometimes required to analyze both fecal coliforms and E. coli. In spite of the fact that 24 h is sufficient time for the determination of E. coli as an indicator of fecal contamination Weiss and Humber, 1988; Feng, 1998; Harrigan, 1998., the test time is 4 days by the standard methods for determination of fecal coliform levels when both fecal coliforms and E. coli analysis are demanded. For routine analysis of foods, time and cost of analysis is obviously very important. Likewise with the MUG method, there are some other rapid meth-

ods for the determination of the bacteriological quality of foods. Some of them are very expensive andror not certified by international quality control organizations while the MUG method is already approved by AOAC Feng, 1998; Hitchins et al., 1998.. The research data presented in this paper suggests that when determining E. coli levels in food samples, EC broth positive tubes should be sub-cultured into 2.5 ml LST broth q MUG instead of TW medium, since this method is more sensitive, cheaper 2.454 vs. 2.887 EUROrsample. and faster 1618 h vs. 610 days. than the conventional ISO method. During routine analysis of foods, 610 days for E. coli analysis is unacceptable. For the routine analysis of E. coli in food industry, LST broth q MUG, as an absencepresence test, the results are obtained within 1618 h. The MUG method for the determination of the presence of E. coli is also an acceptable method in the food industry where there is no chance of applying standard ISO or AOAC methods because of the required longer test time. For the routine analysis of rapidly perishable foods such as pasteurized milk, cream cake etc., if both fecal coliforms and E. coli analyses are required, the described alternative MUG method is advisable.

Acknowledgements This study is a part of the project no. 97111201, entitled AResearch on Fecal Coliforms in FoodsB. The authors thank Ankara University Research Fund for financial support and Dr. E. Baspinar from Ankara University for his kind help in performing the statistical analyses.

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