Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil * Isolamento e identificação sorológica de Escherichia coli enteropatogênica em leite pasteurizado

Correspondence to: Mara LP Queiroz. Disciplina de Microbiologia e Imunologia Departamento de Patologia e Laboratórios, UERJ Av. 28 de Setembro, 87 fundos, 3o andar 20551-030 Rio de Janeiro, RJ, Brazil E-mail: queiroz@uerj.br *Presented on the XIX “Congresso Brasileiro de Microbiologia”, Rio de Janeiro (Brasil), 1997, and in the 98th Meeting of the American Society for Microbiology, Atlanta, Georgia (USA), 1998. Submitted on 29/6/2000. Reviewed on 10/1/2001. Approved on 2/3/2001.


INTRODUCTION
Pasteurized milk in Brazil is classified in the types A, B and C, according to their fat content and to the Brazilian standards of microbiological counts. 12In this study, pasteurized milk samples of the types B and C of three commercial brands were analyzed.The maximum bacterial counts allowed by law are: type B pasteurized milk -mesophilic microorganisms: 8.0 x 10 4 CFU/ml; psychrotrophic and thermoduric microorganisms: 8.0 x 10 3 CFU/ml; total coliforms: 4 MPN/ml, fecal coliforms: 1 MPN/ml; type C pasteurized milk -mesophilic microorganisms: 3.0 x 10 5 CFU/ml; psychrotrophic and thermoduric microorganisms: 3.0 x 10 4 CFU/ml; total coliforms: 10 MPN/ ml and fecal coliforms: 2 MPN/ml.Mesophilic, psychrotrophic, and thermoduric microorganisms count is an indicator of potential spoilage and a means of measuring food quality and safety.Coliform bacteria count in milk and dairy products is an indicator of the sanitary conditions or practices during production, processing and storage of the product. 2,12Recovery and counting of Escherichia coli is used as an index of recent fecal contamination and suggests that other microorganisms of fecal origin, including pathogens, might be present. 15 coli is responsible for several outbreaks of diarrhea in children and adults after ingestion of contaminated water and food, including milk and dairy products.7 Enteropathogenic E. coli (EPEC) have been implicated in food and waterborne human illnesses, especially as an important agent of infantile diarrhea in developing countries.14,15 In Brazil, EPEC strains are recovered from 30% or more of the diarrhea cases in infants of low social economic status living in cities. 10 In the present study the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, was investigated through mesophilic, psychrotrophic and thermoduric microorganism plate counting, counts of total and fecal coliform bacteria and serological identification of EPEC strains among E. coli isolates.

METHODS
Pasteurized milk samples of three different commercial brands were purchased in supermarkets and bakeries in Rio de Janeiro between June 1995 and February 1996.Food samples were acquired in regular consumer packages and immediately transported in ice bath to the laboratory, where they were processed within 2 hours.A total of 90 samples (1.000 ml each) of pasteurized milk of type B (15 samples of each brand) and type C (15 samples of each brand) were examined.
Standard plate and total and fecal coliform counts were performed according to the standard method for dairy products. 2olation and identification of E. coli were carried out using conventional physiological tests. 11,16Serological characterization of EPEC was carried out using slide agglutination method with polyvalent and monovalent antisera.The isolates were first tested with OK polivalent antisera and then with O monovalent antisera for serogroups O26, O55, O111, O119, O114, O125, O142, O158, O86, O126, O127, and O128.The strains belonging to the same serogroups and isolated from the same samples were reported only once.Positive control strains were included in each experiment run.

RESULTS
Ninety milk samples of three commercial brands (I, II and III) that were in accordance with the Health Ministry's National Department of Sanitation Surveillance (Secretaria Nacional de Vigilância Sanitária do Ministério da Saúde -SNVS) 12 were analyzed.
Standard plate counts of mesophilic, psychrotrophic, and thermoduric microorganisms in pasteurized milk samples are summarized in Table 1.Type C milk samples of brand I showed microorganism counting within the SNVS maximum range, while type B milk samples of the same brand presented psychrotrophic and thermoduric counting above the SNVS maximum value, 13.3% and 5.6%, respectively.Types B and C milk samples of brands II and III had counting above the SNVS maximum value to all indicators microorganisms.Total and fecal coliform counts in milk samples are summarized in Figure .The total coliform MPN for types B and C milk samples of brand I were 33.3% and 20% of the samples respectively, above the SNVS maximum value, and fecal coliform MPN were 26% and 20%, respectively.Total coliform MPN were also above the SNVS maximum value for types B and C milk samples of brand II, i.e., 40% and 60%, respectively.For the same samples, fecal coliform MPN levels found were 40% and 53.3%, respectively.Types B and C milk samples of brand III presented a total (73% and 86.6%, respectively) and fecal (53.3% and 60%, respectively) coliform MPN above the regulated levels.
A total of 208 E. coli colonies were isolated from 41.1% of the 90 samples, and 46 (22.1%) strains were serologically identified as EPEC (Table 2).EPEC serogroups were isolated from all milk samples of the three brands.There were found 4 (8.6%) in brand I, 31 (67.3%) in brand II, and 11 (23.9%) in brand III.The most common EPEC serogroup found was O55 (15.2%).Type C milk samples showed the highest rate of EPEC serogroups (58.5%).Thirtyfive (76.08%) of these EPEC strains were isolated from 10 milk samples.EPEC strains were predominantly found in milk samples of brand II (Table 3).

DISCUSSION
The microbiological analysis of milk samples showed a significant deficiency in hygienic and sanitary quality conditions (Table 1 and Figure).Other studies on milk and/or dairy products showed similar results both in Brazil 3,6,9,17 and other countries. 1,5,8t was observed that types B and C milk of brand III had a high coliform level (Figure ) although EPEC strains prevailed in samples of brand II (Table 3).EPEC strains were also found in samples of brand I (Table    3), which showed to have the closest counts above the SNVS maximum value (Table 1 and Figure).It was also found that sample 2 of brand II and sample 3 of brand III presented at the same time microorganisms counting above the SNVS 12 (1997) maximum value, and EPEC serogroups, indicating a high contamination level of this products.
Forty-six (22,1%) out of 208 E. coli colonies isolated were positive for EPEC serogroups.Similar results were obtained by Tibana 17 (1981) when it was found 25.83% of positive EPEC serogroups among 78 of E. coli strains isolated from milk samples in Rio de Janeiro.However, in the city of Ouro Preto, Nascimento et al 13 (1988) isolated 605 strains of E. coli from one type of soft cheese and 9.8% were positive for EPEC serogroups.Frank & Marth 7 (1978), in the United States, did not detected EPEC serogroups in soft and semi-soft cheese samples.Simango 15 (1995) isolated 126 strains of E. coli from several foods and drinks consumed in a rural community, in Harare, Zimbabwe, and 7.5% of them were EPEC serogroups.
In the present investigation all EPEC serogroups were found in the milk samples tested and O55 was the most frequent one (15.2%).The O55 EPEC serogroup is one of most prevalent in children diarrhea in Brazil. 18ecent studies based on virulence properties rather than serological characteristics showed that EPEC serogroups are heterogeneous and although most of strains belonging to these serogroups are actually EPEC, but many of them are not. 4Given that types B and C pasteurized milk are consumed mostly by the poor population in Rio de Janeiro, as well as in many Brazilian cities, due to its lower cost and nutritional value, the results showed that several EPEC serogroups can be easily isolated from pasteurized milk in the community.It is worth to emphasize the importance of surveillance in manufacturing pasteurized milk, since the presence of EPEC possibly indicates enteropathogens contamination.

Figure -
Figure -Enumeration of total and fecal coliforms above the levels permitted by SNVS 12 (1997) in pasteurized milk samples.

Table 1 -
Standard plate counting of mesophilic, psychrotrophic and thermoduric microorganisms in pasteurized milk samples in Rio de Janeiro, Brazil.* www.fsp.usp.br/rspSerological identification of EPEC in milk Silva ZN da et al.

Table 2 -
Distribution of enteropatogenic E. coli serogroups isolated from pasteurized milk samples.

Table 3 -
Distribution of different enteropatogenic E. coli serogroups isolated from pasteurized milk samples.