Prevalence of enteropathogenic and enterotoxigenic Escherichia Coli in foods of animal origin in southern Brazil

Prevalência de Escherichia coli enteropatogênica e enterotoxigênica em alimentos de origem animal no sul do Brasil

PREVALENCE OF ENTEROPATHOGENIC AND ENTEROTOXIGENIC Escherichia coli IN FOODS OF ANIMAL ORIGIN IN SOUTHERN BRAZIL¹

PREVALÊNCIA DE Escherichia coli ENTEROPATOGÊNICA E ENTEROTOXIGÊNICA EM ALIMENTOS DE ORIGEM ANIMAL NO SUL DO BRASIL

José Antonio Aleixo² Gladis Paim Aver³

SUMMARY

Prevalence of Escherichia coli in foods of animal origin from the city of Pelotas, Brazil, was determined. The ocurrence of enterotoxigenic (ETEC) and classic enteropathogenic (EPEC) serogroups among E. coli isolates was determined. E. coli was isolated from 68% of the food samples surveyed. Of 36 food samples tested, 11(30%) and 24(66%) were positive for EPEC and ETEC strains respectively. However, of 187 E. coli isolates tested, 30(16%) were EPEC compared to 75(40%) which were ETEC. The antibiotic resistance pattern revealed that the isolates were highly sensitive to all antibiotics tested.

Key words: Escherichia coli, EPEC, ETEC, foods, Brazil.

RESUMO

Foi determinada a prevalência de Escherichia coli em alimentos de origem animal na cidade de Pelotas, RS. Determinou-se a ocorrência de cepas enterotoxigênicas (ETEC) e enteropatogênicas clássicas (EPEC) entre os isolamentos de E. coli. Em 86% das amostras de alimentos analisadas foi detectada a presença de E. coli. De 36 amostras de alimentos testadas, 11(30%) e 24(66%) foram positivas para EPEC e ETEC, respectivamente. Entre os 187 isolamentos de E. coli testados. 30(16%) pertenciam a sorogrupos de EPEC e 75(40%) foram positivos para ETEC. O perfil de resistência à antibióticos revelou que os isolados foram altamente sensíveis a todos os antibióticos testados.

Palavras-chave: Escherichia coli, EPEC, ETEC, alimentos. Brasil.

INTRODUCTION

Foods contaminated with enteropathogenic bacteria are an important factor contributing to the high incidence of diarrhoea in developing countries with poor sanitary standards. In this respect, classic enteropathogenic and enterotoxigenic strains of Escherichia coli are of particular importance since they were involved in several outbreaks of diarrhoea associated with the consumption of contaminated foods and water (MARRIER et al, 1973; TAYLOR et al, 1983; WOOD et al., 1983a).

The mechanism of EPEC pathogenesis is not well understood but it is known that the bacterium requires a plasmid-encoded adherence factor and chromosome encoded factors to cause alterations in the intestinal epithelial cells (DONNENBERG & KAPPER, 1992). Enterotoxigenic E. coli (ETEC) strains adhere to and colonize the epithelial cells of the small intestine and secrete a heat stable (ST) and/or a heat labile (LT) enterotoxin that causes diarrhoea in humans and animals (LEVINE, 1987).

Several papers have reported the ocurrence of EPEC and ETEC in raw and processed foods and in water in Brazil (REIS et al., 1982; SATO et al, 1983; FRANCO et al, 1985; FRANCO et al, 1987), however most of these studies were done on foods from the Southeast region of the country. There are no data available on the contam ination of foods by enteropathogenic bacteria in Southern Brazil, which is one of the regions with high standards of living in the country. In this paper we report on the ocurrence of EPEC and ETEC in foods of animal origin currently consumed in Pelotas, a medium size city in the extreme South of Brazil.

MATERIALS AND METHODS

A total of 16 samples of pasteurized milk, 10 samples of ground beefand 10 samples of white cheese (Minas type) were examined for the presence of EPEC and ETEC. Milk and meat samples were purchased directiy from food stores. Cheese samples were bought from street vendors at "feiras livres" (open markets), a common way to commercialize foods in most Brazilian cities. Food products were transported to the laboratory in an ice box and cultured within 2h of purchase.

Food samples were initially enriched by mixing 25g or ml of food with 225ml of lauril-sulfate-tryptose broth (LST, Difco) and incubating at 37°C for 24h. Isolation of E. coli from enriched cultures was carried out on eosin-methylene blue agar (EMB-Levine, Difco) incubated at 35°C for 24h. Ten colonies with morphologic characteristics of E. coli were transferred into separate tubes containing triple-sugar-iron agar siants (TSI, Difco) and LST broth with a fermentation tube, and incubated at 35°C for 24h. Colonies that yelded yellow butt and siant, did not produce H₂S in TSI agar, and produced gas in LST broth were further characterized according to EDWARDS & EWING (1972). The E. coli isolates thus obtained were stored at - 20°C in trypitic soy broth containing 15% of glycerol. The isolates were propagated in sheep blood agar for serological, toxin and antibiotic resistance tests.

The production of ST enterotoxin was detected using the suckling mouse assay (DEAN et al, 1972), and the production of LT enterotoxin was detected by indirect hemagglutination of sheep red blood cells (RICCI & PESTANA DE CASTRO, 1986). For EPEC detection the isolates were scree-ned by slide agglutination with three polyvalent O antisera (poly A: O26, O55, O111, O119; poly B: O114, O125, O142, O158; poly C: O86, O126, O127, O128; Probac do Brasil, São Paulo). Isolates that were positive in the screening were then agglutinated with monovalent O antisera. The method of BAUER et al., (1966) was used to determine the resistance of the isolates to the following antimicrobials: amikacin (30ug ), nalidixic acid (30ug), ampicilin (10ug), cephalothin (30ug), chioramphenicol (30ug), trimethoprim-sulfamethoxazole (1,25 + 23,5ug), strep-tomycin (10ug), gentamycin (10ug), nitrofurantoin (30ug), polymixin B (300U), tetracycline (30ug), and tobramycin (10ug).

RESULTS AND DISCUSSION

A total of 36 food samples were examined. The frequency of food samples contaminated with E. coli, EPEC and ETEC is shown in Table 1. The high number of samples positive for E. coli found in this survey was not unexpected since the Brazilian microbiological standards for foods allows the existence of fecal coliforms even in pasteurized milk and cheese (MINISTÉRIO DA SAÚDE, 1987). However, the number of food samples positive for EPEC (30.5%) and ETEC (66.6%) was not anticipated. These pathogens occur in foods throughout the world usually at low frequencies (DANIELSSON et al., 1979; WOOD et al., 1983; ABBAR, 1988; AHMED et al., 1988). In Brazil, both EPEC and ETEC have been detected in foods of animal origin before, though not at the levels detected in this study (REIS et al, 1980; FRANCO et al, 1987). The methodology used for isolation of E. coli in this study did not include enrichment at 44.5°C to prevent loss of plasmids encoding enterotoxin production (HILL & CARLISLE, 1981). However, this fact alone cannot explain the high isolation rates obtained. More than likely our data reflect poor hygienic practices during production of these foods. Milking of cows in the Southern region is usually done manually, under unsanitary conditions, and the milk remains several hours at room temperature before being transported to a processing plant. The type of cheese sampled in this study was made with non-pasteurized milk. The meat carne from animals slaughtered at small facilities lacking proper sanitation.

The number of E. coli and of EPEC and ETEC isolates obtained, EPEC serogroups, ETEC phenotypes is shown in Table 2. The high proportion of EPEC (16%) and ETEC (40%) among the E. coli strains recovered is in disagreement with the relatively low frequencies found by others in different countries. In Brazil, REIS et al. (1980) found only 1.5 % of ETEC among E. coli isolates while FRANCO et al. (1985) reported 0.4% of EPEC and 1.2% of ETEC. WOOD et al. (1983b), in México, found 10% of ETEC in isolates from commercial foods. However, AHMED et al (1988) reported that 33% of the E. coli strains isolated from Egyptian white cheese were enteropathogenic.

Production of enterotoxins was very common among the E. coli strains isolated from all types of foods. The majority of ETEC produced only the ST enterotoxin, although producers of the LT toxin only and of LT and ST simultaneously were also found. This result is in contrast with previous findings in Brazil where LT producer strains were prevalent (REIS et al., 1980; FRANCO et al., 1987).

The EPEC serogroups 026, 055 and 0119 isolated from cheese are relatively frequent in infants with diarrhea in some Brazilian cities (QUEIROZ et al., 1985; GOMES et al., 1991). The other serogroups detected, although frequently isolated from foods (FRANCO et al., 1985; PETRI et al., 1989), are not commonly involved in cases of infant diarrhea. EPEC isolates from serogroups 0114, 055 and 0158 gave positive results in the suckling mouse assay. It seems that positive suckling mouse assay is common among EPEC isolates from developing countries (KORNACKI & MARTH, 1982).

The E. coli strains isolated from foods were sensitive to the antimicrobial agents tested. Our results confirm previous findings in Brazilian foods and water (REIS et al, 1980; SATO et al., 1983) and suggest that, in Brazil, the use of these drugs in medical and veterinary practices did not affect the resistance pattern of E. coli found in foods so far.

The epidemiological significance of these results is not clear at present since foodbome gastroenteritis caused by EPEC or ETEC have not yet been reported in Brazil. However, studies on the prevalence and epidemiology of diarheal diseases in adults that include identification of etiological agents are rare in Brazil. Studies with infants up to the age of 5 years show that EPEC and ETEC are among the most frequently detected enteropathogens (TRABULSI et al., 1985; GOMES et al, 1991). Moreover, foodbome gastroenteritis caused by enteropathogenic strains of E. coli is relatively common all over the world, even in industrialized countries (MARRIER et al, 1973; DANIELSSON et al., 1979; TAYLOR et al, 1983). Thus, it is probable that foods of animal origin play an important role in the dissemination of EPEC and ETEC in our community.

ACKNOWLEDGMENT

The technical assistance of Ms. Alegani V. Monteiro is greatly appreciated.

² Médico Veterinário, Professor Titular, Departamento de Nutrição, Universidade Federal de Pelotas (UFPel), 96010-900, Pelotas, RS. Autor para correspondência.

³ Farmacêutico-Bioquímico, Professor Assistente do Departamento de Microbiologia e Parasitologia da UFPel.

Recebido para publicação em 01.11.95. Aprovado em 21.02.96.

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¹

Trabalho realizado com auxílio da FAPERGS.

Publication Dates

• Publication in this collection
  25 Sept 2008
• Date of issue
  Aug 1996

History

• Received
  01 Nov 1995
• Accepted
  21 Feb 1996