Biochemical and Physiological Characteristics of Escherichia coli isolated from Different Sources

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1 Biochemical and Physiological Characteristics of Escherichia coli isolated from Different Sources Sherfi S., A.1, Dirar, H., A.2, Ibrahim F. Ahmed3 1 Department of Basic Sciences, Faculty of Medical Laboratories Sciences, International University of Africa, Khartoum, Sudan. 2 Department of Botany andagricultural Biotechnology, Faculty of Agriculture, University of Khartoum, Khartoum, Sudan. 3 Dean of the Faculty of Medical Laboratories Sciences, International University of Africa, Khartoum, Sudan. ABSTRACT In this study ninety four isolates of Escherichia coli were obtained from different sources of water, sewage, and human and animal feces by using multiple tube fermentation technique or by direct streaking on MacConkey agar. All isolates were first identified biochemically using a Japanese API like system. The physiological characteristics of the E. coli isolates were examined by studying their growth in different culture media, i.e., E. coli medium (EC), brilliant green bile broth (BGB), MacConkey broth, and lauryl sulphate tryptose broth, at different temperatures, viz., 30, 37 and 44.5 C. The results of these tests showed that EC broth medium yielded the highest growth density of E. coli at all temperatures. Brilliant green bile broth gave the second best growth density, followed by MacConkey broth, then lauryl sulphate tryptose broth. 146

2 The incubation Temperature 37 C gave the highest growth density in all four growth media, the lowest growth density being obtained at 44.5 C. The results of this study indicated that E. coli can be used as a fecal indicator for contaminated water in Sudan but the choice of media used as well as the survival of the organism in pure water should be taken into consideration. Key words: E. coli, Fecal indicator 1. INTRODUCTION Escherichia coli is inhabitant of the intestinal tract of humans and animals. For this reason scientists argued that if E. coli was found in any habitat other than the intestine, that habitat must have been somehow contaminated with human or animal feces. This led to the suggestion that E. coli should be used as an indicator of the contamination of foods, particularly of water, with human excreta, hence, possible with pathogens (Clark, J. A. and Pagel, J. E. 1977). The test has been developed in temperate countries. The later application of the test in the Third World meant its application in a new environment such as tropical countries whose inhabitants eat different kinds of foods not necessarily made of wheat or potatoes as in cold countries. It is now well known, even in general microbiology (Tortora et al. 1992; Madigan et al. 1997) that the gut flora in humans can change in response to an altered environment and can vary qualitatively, and quantitatively depending on the diet. 147

3 A great amount of research data has questioned the suitability of E. coli as an indicator of fecal contamination of tropical water. Some authors have argued that Vibrio cholerae is a natural inhabitant of tropical waters and the absence of E. coli in this water does not show that the cholera organism is not present (Perez-Rosas and Hazen, 1989). Others showed that pathogens like Salmonella survived for a longer time in tropical waters and that E. coli might not be suitable as indicator of recent contamination (Jimenez et al.1989). It has even been suggested that E. coli is a normal inhabitant of tropical waters and therefore is unsuitable as a fecal indicator (WHO 1995). These are just some examples of research carried out in tropical countries. The Sudan has recently published, through Sudan Standards and Metrology Organization (SSMO), its first preliminary microbiological standards for foods. Naturally, E. coli is adopted in these standards as the major indicator of fecal contamination as generally recommended by the World Health Organization (WHO, 1964) and other international institutions. But Sudan is a tropical country with temperatures soaring up to near 50 C or more in the summer. The major diet of the general rural population is sorghum and pearl millet. It is likely that the micro- flora of the intestines of humans and animals and the survival of E. coli in natural waters is different in this country from Europe and North America. The workers of Sudan preliminary microbiological standards of foods were very much aware of this and wrote: SSMO should sponsor and 148

4 encourage research on the microbiology of foods as related to food standards and safety. This research has been carried out to see if E. coli is a suitable indicator of fecal contamination of food. The strategy to do this is to isolate, characterize and study the physiology of E. coli from different water sources and human and animal feces in Khartoum State where practically all Sudanese communities are represented. 2. MATERIALS AND METHODS 2:1 Collection of samples: - Samples from different sources such as human and animal feces and different types of water (drinking water from zeer, stagnant rain water, irrigation water, and sewage) were collected and used to obtain 94 suspected E. coli isolates. The samples were collected in clean and sterile screw- capped bottles or test tubes from different sites of Khartoum State. The bottles and test tubes were placed in ice. Samples were examined on the same day and those which were not processed within one hour after collection were stored at zero º C for a maximum of two days. 2:2 Isolation of E. coli: - 2:2:1 Isolation from feces: - E. coli isolates from human and animal feces were obtained by direct streaking of the sample on MacConkey, s agar (Mast Diagnostic U.K.) as they needed no enrichment because of their large number. The 149

5 plates were incubated at 37ºC aerobically for 24hrs. All plates were inspected for growth and colonial morphology. The isolates were subjected to confirmation tests on Eosin Methylene Blue (EMB) medium. 2:2:2 Isolation from water and sewage: - Standard multiple tube fermentation technique described by Geldreich (1975) was used for water examination, which is divided into three stages:- presumptive test on Lauryl sulphate tryptose broth (LST), confirmed test on EC medium and completed test on Eosin Methylene Blue (EMB) agar. Typical E. coli colonies picked from EMB agar including isolates from feces were transferred to nutrient agar plates to remove their colors. The Gram-stain was performed on the new growth; E. coli appeared as Gram-negative, short rods occurring singly or in pairs. 2:3 Biochemical identification of E. coli: - All 94 cultures appearing as Gram- negative, short rods or coccobacilli, were subjected to various biochemical tests (special Japanese scheme similar to the API system) (Colle et al., 1996). This biochemical scheme includes triple- sugar- iron (TSI) test, motility test, indole test, urease test, lysine decomposition test, Vogesproskauer (VP) test, and DNAse production test. All media and reagents used in these tests were prepared in the laboratory (Health Laboratory, Khatoum State). Based on the outcome of the tests, each 150

6 isolate was given a code number consisting of four digits, e. g., The digits were determined as follows: code number of lactose represented the first digit, the summation of the (TSI) codes represented the second digit, the summation of lysine, indole and motility codes represented the third digit and the summation of Urea, (V-P) and DNAse codes represented the fourth digit (Colle et al., 1996). Isolates scoring the same code number represent the same strain. 2:5 Physiological tests: - 2:5:1 Effect of the culture media and the incubation temperature on the growth of E. coli:- The media selected were those commonly used in the examination of water for E. coli. One loopful of hrs culture (standard loop; a loop of known dimension -1/400 ml-) of each identified ninety four isolates of E. coli was used to inoculate 10 ml nutrient broth and incubated at 37 º C for 24 hours. One loopful of this culture was inoculated into 10 ml of different types of media, viz., lauryl sulphate tryptose broth (LST), EC medium, MacConkey, s broth and brilliant green bile broth. The media were incubated at different temperatures, i.e., 30 º C, 37 º C, and 44.5 º C for 24 hours. The degree of growth was measured by turbidometric method at the visible range of the spectrum at average wavelength 557 nm, using Spectrophotometer Model 6300 (Jenway Ltd., U.K.) to determine the optical density of the culture, using sterile medium as control. 151

7 3. RESULTS AND DISCUSSION 3:1 Isolation of E. coli:- E. coli isolated from human and animal feces gave smooth pink colonies on the solid isolation medium MacConkey s agar, indicating lactose fermentation. Lactose is included in this medium as a fermentable carbohydrate together with a ph indicator, usually neutral red. Strong acid producers, like Escherichia, Klebsiella, and Enterobacter produced red colonies on this solid medium (Adams and Moss, 2000), whereas those isolated from water sources and sewage produced gas in the liquid enrichment EC medium at 44.5 C. Gas production among identically prepared subcultures varies in amounts from sample to other in the multiple tube tests. Meadows et al. (1990) referred this variability to the erroneous interpretation of the results of coliform multiple tube tests or the growth of E. coli without gas. This has been interpreted as the chance cultivation of anaerogenic or environmentally damaged strains. Meadows et al. (1990) reported that good gas production was obtained in buffered media. Their confirmed test by streaking on EMB agar produced the darkcentered colonies with metallic green sheen. EMB agar is a popular selective and differential medium. The aniline dyes, eosin and methylene blue, are the selective agents but also serve as an indicator for lactose fermentation by forming a precipitate at low ph. Strong lactose fermenters produce green black colonies with a metallic sheen (Adams and Moss, 2000). 152

8 3:3 Identification of E. coli:- 3:3:1 Biochemical tests:- Results obtained from the biochemical tests differentiate five groups of E. coli which differ in (1) gas production from glucose fermentation (2) lysine decarboxylation (3) indole production, and (4) motility test. As shown in Tables 1 to 5, the code numbers (see Material and Methods) obtained for our isolates were: 1370 (61.7%; 58/ 94), 1360 (28.7%; 27/ 94), 1330 (5.3%; 5/ 94), 1170 (3.2%; 3/ 94) and 1350 (1.1%; 1/ 94). Thus the biochemical tests grouped our E. coli isolates into five groups.. (i) Triple sugar iron (TSI): All isolates of E. coli showed a yellow butt and a yellow slope to indicate the fermentation of lactose, sucrose and possibly glucose. The bubbles in the medium indicate gas production from glucose fermentation. About 96.8% of E. coli isolates produced gas from glucose fermentation and gave code number of (2) and about 3.2% isolates did not produce gas from sugar fermentation, and were given code number of (0). It means that most isolates of E. coli produced gas from glucose fermentation and a few of them did not. All isolates obtained from water sources and sewage showed a positive gas production from TSI whereas those obtained from animal and human feces showed 89.47% and 97.44% positive gas production, respectively. We thus notice that gas production from glucose fermentation in E. coli was affected by the source of isolate. This may be related to the nutrients or oxygen available in the source. Farmer et 153

9 al. (1985) reported that 95% of E. coli isolates produced gas from glucose fermentation, % of normal E. coli and 0-10% of inactive E. coli produced gas from fermentation. All isolates showed no blackening along the stab line or throughout the medium indicating no production of H 2 S and were given a code number of (0). (ii) Lysine test: About 64.9% of E. coli isolates showed violet color throughout the medium as positive result of lysine decarboxylation to the corresponding amine with the liberation of carbon dioxide, and were given code number of (1). About 35.1% of E. coli isolates kept the yellow color of media as it was as a negative result of lysine decarboxylation and were given (0) as a code number. Human feces isolates gave 38.5% negative lysine decarboxylation, animal feces isolates gave 36.8%, water source isolate gave 32.0% and isolates obtained from sewage gave 27.3%. Farmer et al., (1985) reported that 90% of E. coli isolates decarboxylated lysine, % of normal E. coli and % of inactive E. coli gave positive lysine decarboxylation. (iii) Urease test The positive result of urease test is indicated by a change in the color of the indicator to red pink. All isolates of E. coli did not change the color of the medium to red pink; all E. coli strains did not produce urease (negative urease) and were given a code number of (0). 154

10 (iv) Indole production: About 93.6% of E. coli isolates gave a red color in the upper layer of the medium, indicating a positive result for indole production and were given a code number of (2), while 6.4% of isolates gave negative results of indole production, and were given the code number (1). Isolates obtained from sewage source showed high percentage of positive indole production up to 100.0%, whereas human and animal feces isolates gave almost equal percentages, 94.87% and 94.74%, respectively, and isolates obtained from water gave 88.0% indole positive. The results obtained from this test showed that indole production in E. coli varied, as reported by Jay (1992). The IMViC reaction designates E. coli type 1 and E. coli type 2 are Water samples contain more of E. coli type 2 than feces and sewage samples. Farmer et al (1985) reported that 98% of E. coli were indole positive, % of normal E. coli and 75-89% of inactive E. coli were indole positive. (v) Voges Proskauer test: All isolates showed a negative reaction towards V P test. No pink color was formed during the test which means that the isolates did not form acetoin. A code number of (0) was given. (vi) Motility: About 94.7% of E. coli isolates were motile showing a turbidity throughout the medium and were given a code number of (4), whereas 155

11 5.3% were non-motile, producing growth only along the line of inoculation, and were given a code number of (2). Isolates of E. coli obtained from water sources gave 92.0% motile. Human and animal feces isolates gave and 94.74%, respectively, and all isolates of E. coli obtained from sewage gave 100.0% motile, similar to that of indole positive results. We notice that all indole positive isolates were also motile except SW 8 which had a code number of 1350 identified as E. coli according to code sheet Table 1 Appendix. That means indole production is correlated with the motility of E. coli. This disagreed with Krieg (1984) who reported that % of E. coli strains were indole positive, whereas 76-89% of E. coli strains were motile. Colle et al., (1996) reported that at least 80% of E. coli strains were motile, though sometimes only weakly, on primary isolation. Contrasting with the typical, biochemically active types, some strains are lactose non-fermenting, non-motile, anaerogenic and biochemcally inactive, like Shigella. Farmer et al., (1985) reported that 95% of E. (vii) DNAse test: DNAse- producing colonies growing on DNA medium were surrounded by clear areas, indicating DNA hydrolysis, while unhydrolyzed DNA is precipitated by added HCl acid. All isolates of E. coli did not produce DNAse and their colonies were not surrounded 156

12 by clear areas indicating absence of hydrolyzed DNA, and were given a code number of (0). Table 1: The confirmatory biochemical tests of E. coli isolates obtained from sewage. (numbers are codes given according to the Japanese system. See text for explanation) Tsi = Triple sugar- iron Suc = Sucrose fermentation Gas = Gas production from glucose H 2 S = production of H 2 S DNA = DNAse test Lysine = Lysine test V. P =Voges proskauer test Urea = Urease test 157

13 Table 2: The confirmatory biochemical tests of E. coli isolates obtained from human feces (first batch) 158

14 Table 3: The confirmatory biochemical tests of E. coli isolates obtained from human feces (second batch) 159

15 Table 4: The confirmatory biochemical tests of E. coli isolates obtained from different sources of water 160

16 Table 5: The confirmatory biochemical tests of E. coli isolates obtained from animal feces 3:4 Physiological tests:- 3:4:1 The effect of culture media and incubation temperature on the growth of E. coli:- The extent of growth for each of the 94 isolates in different media at different temperatures is given as optical density. These values are presented as average values in Figs 1-7. As shown in Figs 1-3 EC medium yielded the highest growth density of all isolates of E. coli at the three temperature degrees of 30, 37 and 44.5 C. Isolates of E. coli obtained from human feces and sewage showed higher growth densities than isolates obtained from water and 161

17 animal feces. Brilliant green bile broth (BGB) medium gave high growth density at different temperature degrees for all isolates of E. coli obtained from the different sources. On this medium isolates of E. coli obtained from sewage and water sources showed higher growth densities than those isolated from human and animal feces. MacConkey broth gave a low growth density of all isolates of E. coli at different temperature degrees compared with EC and BGB media, on which isolates of E. coli obtained from human feces showed highest density of growth than isolates obtained from sewage, water sources or animal feces. LST medium yielded the lowest growth density of E. coli at all temperature degrees tested. E. coli isolates obtained from water sources gave higher growth density than E. coli isolates obtained from human feces, animal feces or sewage. With respect to the effect of growth medium on the growth density of E. coli, our results showed that EC medium yielded the highest growth of E. coli. This may be due to its content of buffers (K 2 HPO 4 and KH 2 PO 4 ) and the bile salts No.3 and tryptose instead of peptone. Hajna and Perry (1943) reported that the EC medium was a highly buffered lactose broth modified by the addition of 0.15% of Difco Bacto bile salts No. 3 and the substitution of Bactotryptose for Bacto peptone. The virtue of the bile salts mixture lies in its enhancement of the growth of coliform bacteria, and its ability to inhibit more or less completely the growth of fecal streptococci and spore formers. The EC medium can be used equally well for the isolation of coliform bacteria at 37 º C or for the isolation of E. coli at 45.5 º C. It can be used 162

18 either as a primary medium for the growth of E. coli or as a satisfactory secondary medium for the confirmation of E. coli. The specificity of the EC medium is 100 percent. The superiority of EC medium for fecal coliforms was also reported by Jay (1986). Colle et al., (1996) reported that entero- hemorrhagic E. coli (EHEC) strains would not grow at 44.5 C in the standard EC medium formulation, but would grow when the bile salts content in the medium was reduced from 0.15% to 0.112%. Brilliant green bile broth gave high growth of E. coli but less than did EC medium. This lower growth may be due to the substitution of lactose with glucose, also bile salts No.3 with dried bovine bile in BGB medium. Black and Klinger (1936) found that brilliant green bile broth did not perform uniformly with all strains of E. coli. MacConkey broth gave a growth rate less than that of BGB medium, in which peptone was used instead of tryptose and there are no buffers in its content. This result agreed with WHO (1995) who reported that MacConkey agar was not strongly selective and would support the growth of a number of non- Enterobacteriaceae including Gram positives such as enterococci and staphylococci. Also Grabow and Du Preez (1979) found that the lowest counts of E. coli were usually recorded on MacConkey s agar. Lauryl sulphate tryptose broth yielded the lowest growth rate of E. coli. This may be due to the absence of bile salt in its ingredients. Hajna and Perry (1943) found that the specificity of lauryl sulphate tryptose broth was 98.7% compared with 100% specificity of EC medium. 163

19 The growth of E. coli at different temperature degree was influenced by the source of E. coli isolate and the type of the growth medium. As shown in Fig. 1, all isolates of E. coli gave highest growth density at 30 C in EC. Isolates obtained from human feces and sewage gave highest growth density followed by the isolates obtained from the water source, whereas the animal feces isolates gave the lowest growth density of E. coli. Fig. 2 shows that at 37 C human feces and sewage isolates enumerated in EC medium yielded the highest growth density, whereas human feces and sewage isolates cultured on LST medium yielded the lowest growth density. Fig. 3 shows that at 44.5 C, human isolates and sewage isolates in EC medium gave the highest growth density, whereas in LST medium human feces and sewage isolates gave the lowest growth density. As shown in Figs. 4 to 7 the effect of the different temperatures on the growth of all isolates of E. coli shows that the temperature degree 37 C gave the best growth density for the isolates of E. coli obtained from animal feces and sewage enumerated on all growth media used in this experiment. Incubation at 30 C gave the best or the same growth density for isolates of E. coli obtained from water or human feces, whereas 44.5 C showed a weak growth for all isolates on different growth media. Results obtained in this experiment show that E. coli isolates gave best growth densities at 37 C, a temperature of incubation recommended by WHO (1995). E. coli is a typical mesophile growing from 7 C- 10 C up to 50 C with an optimum around 37 C. According to Bergey s 164

20 Manual (Krieg, 1984) and (Colle et al., 1996), the optimal temperature of E. coli is 36 C-37 C, though growth occurs over a fairly wide temperature range (18-44 C). The growth rate of E. coli at 30 C was less than that at 37 C, whereas 44.5 C gave the lowest growth rate. This may be due to E. coli being a mesophilic bacterium or due to the tropical climate of Sudan in which temperature degrees are higher than that in Western countries. Similar results were reported by Leroi et al. (1994) although 32 C and 42 C are symmetric around the temperature 37 C at which E. coli had been propagated, relative to 37 C, 42 C reduces both maximal growth rate and yield, whereas 32 C reduces only the former. The temperature 42 C is approximately 0.5 C off the critical thermal limit for extinction of the bacterial population under standard culture conditions, whereas 32 C is more than 10 C away from both the upper and lower thermal limits. Whereas 42 C induces expression of stress proteins that mediate a protective response, 32 C does not. Raina et al. (1995) reported that in E. coli the classical heat shock response appears as a transient acceleration in the synthesis of 20 proteins. APHA (1937) found that greater numbers of E. coli were detected with lactose broth at 37 C over the Eijkman medium at 46 C. 165

21 Fig. 1 The effect of culture medium on the growth of E. coli isolated from different sources at 30 C s ity n e l d a1 tic p O SA SH SS SW Different sources of E. coli LST EC Mac BGB SA: samples obtained from animals feces SH: samples obtained from human feces Ss: samples obtained from sewage Sw: samples obtained from water 166

22 Fig. 2 The effect of culture medium on the growth of E. coli isolated from different sources at 37 C ity n s e a l d tic p O SA SH SS SW Different sources of E. coli LST EC Mac BGB 167

23 Fig 3 The effect of culture medium on the growth of E. coli isolated from different sources at 44.5 C ity n s e1 d l a 0.5 tic p O 0 SA SH SS SW Different sourc es of E. coli LST EC Mac BGB Fig. 4 The effect of incubation temperature on the growth of E. coli isolated from water sources ity 1.68 n s e1.67 l d a1.66 tic p 1.65 O C 37C 44.5C Temperature Fig. 5 The effect of incubation temperature on the growth of E. coli isolated from sewage 168

24 ity s n e l d tica p O C 37C 44.5C Temperature Fig. 6 The effect of incubation temperature on the growth of E. coli isolated from human feces ity s 1.67 n e l d a 1.64 p tic o C 37C 44.5C Temperature 169

25 Fig. 7 The effect of incubation temperature on the growth of E. coli isolated form the animal feces 170

26 CONCLUSION The results obtained from this research show that E. coli can be used as fecal indicator of water contamination in Sudan. But some reservations should be taken into consideration: - EC medium gave best growth density of E. coli than Mac, LST and BGB. - The biochemical tests differentiated E. coli isolates into five groups. - Some isolates of E. coli does not produce indole also some were not motile and others do not produce gas from glucose fermentation. - Indole production in E. coli is linked to motility. - Growth of E. coli at 44.5 C is very weak. 171

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