María Laura Arias1 Rafael Monge-RojaS2 Carolina Chaves1 Florencia Antillón1
Received 19-VI-2000. Corrected 24-X-2000. Accepted 27-X-2000.
Abstract:
Escherichia coli 0157: H7 has emerged as a new pathogen and is found worldwide. We studied the effect of several storage temperatures on the survival of this bacterium in common foods from a neotropical environment (Costa Rica) because at least seven clinical cases have been reported from the country, and no epidemiological link or probable food association has been described. High (106- 108 CFU/ml) and low (104-106 CFU/ml) populations of E. coli were inoculated (three replications) in ground meat, chopped cabbage, chicken giblets and pasteurized milk and incubated at 0, 6 and 12°C for 24, 48 and 72 h. Vegetables and milk were also stored at 22°C for the sarne periods.The E. coli 0157: H7 enumeration was done according to the methodology describes in the Bacteriological Analytical Manual. Populations of E. coli 0157: H7 showed either an increasing or decreasing trend, according to temperature, time or food base. Our data indicate that E. coli 0157: H7 is capable of surviving and growing in meat, cabbage, milk and chicken giblets; food items commonly consunied by Costa Ricans.
Key words
Escherichia coli 0157: H7, meat, milk, cabbage, chicken giblets, survival.
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The intestinal tract of ruminants is its prime reservoir. Therefore, food of animal origin has been linked as a vehicle. Nevertheless, this bacterium has also been associated with illness caused by dairy products, vegetables, salads, apple juice and even water (Wilshaw et al. 1994). Person to person spread also occurs.
Recovery frequency from food is low (Sekla et al. 1990), as is the contamination levels of food associated with outbreaks (10-6200 CFU/g) (Todd et al. 1988). At the sarne time, its infecting dose has been described as low, in some cases, two cells / 25 g of food have been known to cause illness (Sekla et al. 1990) and a high acidic tolerance has also been described (Wilkinson 1997). Strict commercial regulations have been developed worldwide, including a zero tolerance level for food imported by the US (Anonymous 1997).
Human infection with E. coli 0157 has been reported from over 30 countries in six continents. Annual incidence rates of 8 per 100 000 population or greater have been described from the USA, Canada and Argentina, in regions characterized for having four seasons. Developing countries, especially those from the tropical belt of the planet, have climatic conditions that favor the proliferation of contaminating bacteria, a fact reflected in the high prevalence of diarrheic diseases, thus, the incidence of E. coli 0157: H7 should be higher in the tropics than in temperate regions. Maybe because of the recent emergence of tus agent, there are almost no reports of its isolation in tropical countries. In Costa Rica at least seven clinical cases in children, two of them fatal, have been reported, but no epidemiological link or food association has been described (Herrera 1998). There are no reports from the country evaluatig E. coli 0157: H7 in risky food: it is vital to evaluate bacterial behavior at different storage conditions, to minimize transmission by food.
The purpose of this study was to detemine the effect of different storage temperaturas on the survival of E. coli 0157: H7 in milk, beef, vegetables and chicken giblets.
Materials and Methods
Foods: Meat, pasteurized milk, packaged chopped cabbage and chicken giblets were purchased from a retail grocer in San José, Costa Rica. All foods were kept at 2 to 5°C between the time of purchase and initiation of experiments, which never exceeded 4 h. Meat was chopped aseptically to avoid the great bacterial charge present in ground meat and standardizing the size and extent of variables.
Inoculum preparation: A human isolated strain of E. coli 0157: H7 was used. Stock cultures were maintained at -70°C on tryptic soy agar slants (Oxoid) and acúvated in tryptic soy broth (pH 7.0) at 35°C. Culture was transferred by loop inocula twice at 24 h intervals to 100 ml of tryptic soy broth in 250 ml Erlenmeyer flasks. The last culture was considered as the large-population suspension (106 to 108 CFU/ml) of E. coli 0157: H7 for inoculating test foods. A low-population suspension (104-106 CFUml) was prepared by adding 1 ml of the large-population suspension to 99 ml of tryptic soy broth.
Procedure for inoculating foods: Approximately 1 kg of ground meat, chopped cabbage and chicken giblets and 1 L of pasteurized milk was separately placed in a sterile poIyethylene bag and inoculated with 50 ml of large-population suspension of enterohaemorrbagic E. Coli. Each food was homogenized in a Stomacher for 2 min. Immediately, three 200 g (or ml) samples of each food were taken and incubated at 0, 6 and 12°C for 24, 48 and 72 h. Vegetables and milk were also stored at 22°C for the same periods. A 25g sample of each food was taken before incubation in order to perform an initial count of E. coli 0157: H7. ]]>
The same procedure was used with another 1 000 g of ground meat, chopped cabbage and chicken giblets and another 1 000 ml of pasteurized milk inoculated with 50 ml of small-population suspension.Three independent trials were performed for each food. For each trial, a non-inoculated sample was used as control.
Procedures for enumeration of E. coli 0157: H7: Immediately after each incubation period, 25 g samples were taken and combined with 225 ml of sterile peptonated water 0. 1 % in a sterile polyethylene bag and pummeled with a Stomacher for 2 min. Wash fluid was serially (1: 10) diluted and surface plated (0,1 ml) on duplicate sorbitol McConkey agar (SMA) (Oxoid). The SMA plates were incubated at 35°C for 20 to 22 h before colonies of E. coli 0157: H7 were counted. The same procedure was used with the control samples (Anonymous 1995).
pH measurements: The pH of the primary diluent in which food samples were pummeled was measured at each microbiological analysis.
Statistical analysis: A Duncan multiple range test was used. Each value represents the mean of six values (duplicate values for each sample analyzed from three independent trials).
Results
Meat: Populations of E. coli 0157: H7 on ground meat stored at 0, 6 and 12°C increased significantly
(p< 0.05) during the fírst 48 hours, whereas the populations decreased around 1 logarithm within 72 hours at the same storage conditions (Fig. 1). The averaged pH showed a slight drop during the storage period (6.1 to 5.4) ]]>
Milk: Unlike the studies of ground meat, populations of E coli 0157: H7 declined significandy (p<0.05) within 24 hours when milk inifially containing 108 CFU/ml was kept at 0 and 6°C. However, increases were detected within 48 hours of storage at 6 and 12°C (Fig. 2). Within 72 hours of incubation at 0 and 6°C, a significant decrease of E coli 0157: H7 was detected in milk samples containing a low-population inoculum. On the contrary, populations of E. coli 0157: H7 on milk stored at 12 and 22°C increased significantly (p<0.05) after 48 h.
Cabbage: Populations of E. coli 0157: H7 decreased significantly on chopped cabbage stored at 22°C after 24 hours, decreasing 3 log within 72 hours. An increase after 48 h of storage at 6°C was followed by significant (p<0.05) decrease at 72 h in the chopped cabbage initially containing a large inoculum (108 CFU/g). On the other hand, populations on cabbage stored at 12°C increased significantly within 72 h. A significant increase (p<0.05) of E coli population was detected on chopped cabbage initially containing a low inoculum (105 CFU/g) after 72 h of storage at O°C. On the contrary, a significant decrease was observed in cabbage stored at 6 and 12°C for 72 h (Fig. 3). The shift in pH at 0, 6 and 12°C ranged between 7.5 to 6.0; the most marked rate of decrease in pH occurred in vegetables stored at 22°C (7.5 to 5.3).
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Chicken giblets: Populations of E. coli 0157: H7 decreased significantly in chicken giblets stored at 0, 6 and 12°C for 24 h, in samples initially containing low and large inoculum. Within 48 h incubation of storage at 12°C a significant increase of E. coli 0157: H7 was detected in chicken giblets initially
inoculated with a large population. A similar behavior was observed in chicken giblets containing low inoculum within 48 h of storage at O°C. Populations in low and large inoculum samples stored at O°C declined significantly after 48h (Fig. 4). The pH shifted between 6.5-5.9.
Discussion ]]>
Foods of animal origin have been described as primary sources of enterohaemorrhagic Escherichia coli (EHEC) infections (AbdulRaouf et al. 1993) and meat has been describes as the principal vehicle of transmission of this bacterium to human beings (Szaho et al. 1990, Roberts et al. 1995). E. coli 0157: H7 has been isolated from dairy cattle, calves, chickens, swine and even from sheep, and from their retail meat (Doyle and Schoeni 1987, Merimelstein 1993, An-Hung et al. 1995). Several authors have described its survival in ground beef during frozen storage (Doyle and Schoeni 1987). Our results coincide with these observations, independently of the population inoculated to meat, or the storage temperature, the numbers of E. coli increased significantly (p<0.05) during the first 48 hours of incubation.Our results also show survival and multiplicative capability in chicken giblets stored at 0, 6 and 12°C. Griffin and Tauxe (1991) did not recover this bacterium from raw chicken, but our results show that contaminated or cross contaminated chicken giblets may harbor important numbers.
Consumption of unpasteurized milk has also been associated with E. coli 0157: H7 outbreaks (Borczyk et al. 1987, Bielaszewska et al. 1997). Behavior of this bacterium at several incubation temperatures and times shows important resistance to low temperaturas, and multiplication especially when stored at 12 and 22°C; the shift in pH also failed to inhibit growth. These results paralel those of Massa et al. (1999) who report increase in the viable population of EHEC inoculated into milk and stored at 8°C.
On the other hand, our results, like those of Ruscisa and Sobol (1995) show that E coli 0157: H7 can grow on refrigerated packed vegetables, as has been reported for Listeriamonocytogenes (Berrang et aL 1989, Beuchat and Bracket 1990, Monge and Arias 1997), Salmonella (Rosas et al. 1984) and Aeromonas hydrophila (Berrant et al. 1989). During the first 24 hours, E. coli incubated at 0, 6 and 12'C showed an increase in number, nevertheless, at 22°C it presented a significant decrease in numbers probably due to competition and the important pH change (7,5-5,3). The decrease in pH can be attributed to the fermentative capability of this microorganism and to competition, including lactic bacteria. Similar results have been obtained by Faith et al. (1997) who found reductions in number of bacteria of 2-4 log 10 CFU/g when stored in pepperoni at 21°C for 14 days.
Vegetables and fruits can become contaminated through cattle manure used in soil or through the use of contaminated water for irrigation or washing (Cieslat et al. 1993). At the same time, usual maintenance of packed vegetables is at refrigeration temperatures (8-10°C), that have no effect on this bacterium. Richert et al. (2 000) report similar results, since they observed survival of EHEC on produce held at 4°C and growth at 15°C.
A recent outbreak of enterohaemorrhagic infection in kindergarten children from Japan is evidence of bacterial survival and multiplication on vegetables. The contaminated food was identified as a potato salad (Makino et al. 1997).
Increased attention to hygiene would be useful, but better risk reduction can be achieved through controlling points of potential contamination in the field and during the processing and handling of the products.
Findings made in this study indicate that E. coli 0157: H7 is capable of surviving and growing in common food: meat, cabbage, milk and chicken giblets under the conditions that are normal in tropical countries such as Costa Rica.
Acknowledgements ]]>
We thank Laura Villalobos for her cooperation, W. Bussing for correcting the English grammar and several anonymous reviewers for their useful comments. This work received support from Oxoid and the Vice President of Research, Universidad de Costa Rica, project 430-99-214.Resumen
Escherichia coli 0157, H7 ha emergido como un nuevo patógeno y se encuentra mundialmente. En Costa Rica, por lo menos ha informado de siete casos clínicos, y no se ha encontrado un lazo epidemiológico o asociación con alimentos. El propósito de este estudio fue determinar el efecto de diferentes temperaturas de almacenamiento sobre la sobrevivencia de esta bacteria en alimentos de uso común. Una población alta de E. Coli (106- 108 UFC/ml) y una baja (104-106 UFC/ml) fueron inoculadas (tres réplicas) en carne picada, repollo picado, vísceras de pollo y leche pasteurizada e incubadas a 0, 6 y I°C por 24, 48 y 72 h. Los vegetales y la leche también fueron incubados a 22°C por los mismos períodos. La enumeración de E. Coli 0157: H7 se realizó de acuerdo a la metodología descrita en el Bacteriological Analytical Manual. Las poblaciones de E. coli mostraron tendencias a aumentar o disminuir, dependiendo de la temperatura, tiempo y base alimenticia. Nuestros datos indican que la E. coli 0157: H7 es capaz de sobrevivir y crecer en carne, repollo, leche y vísceras de pollo, alimentos de uso común en Costa Rica.
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1Facultad de Microbiología, Universidad de Costa Rica, 2060 San José, Costa Rica, fax (506) 207 5440, mlarias@cariari.ucr.ac.cr
2 Instituto Costarricense de Investigación y Enseñanza en Nutrición y Salud, Aptd 4-2250, Tres Ríos. ]]>
