Prolonged fecal shedding of Shiga toxin-producing Escherichia coli among children attending day-care centers in Argentina

E. Miliwebsky^1*, N. Deza¹, I. Chinen¹, E. Martínez Espinosa¹, D. Gómez², E. Pedroni³, L. Caprile ⁴, A. Bashckier¹, E. Manfredi^1, G. Leotta¹, M. Rivas¹

¹Servicio Fisiopatogenia, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr. Carlos G. Malbrán", Av. Vélez Sarsfield 563 (1281) Buenos Aires;
²Laboratorio de Bacteriología, Instituto Nacional de Epidemiología "Dr. Juan H. Jara" - ANLIS "Dr. Carlos G. Malbrán" Mar del Plata;
³Dirección de Epidemiología de la Provincia de Entre Ríos;
⁴Laboratorio de Bacteriología, Hospital Provincial de Rosario, Argentina.

*Correspondencia. E-mail: emiliwebsky@anlis.gov.ar

ABSTRACT

In this report we describe the detection and duration of fecal shedding of Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 in symptomatic and asymptomatic cases during four events occurred among children in day-care centers in Argentina. In each event, the cases were identified among children, family contacts and staff members of the Institution. The isolates were characterized by pheno-genotyping and subtyping methods. The STEC fecal shedding was prolonged and intermittent. Strains O157:H7 (1^st event); O26:H11 (2^nd event); O26:H11 (3^rd event) and O145:NM (4^th event) were shed during 23-30, 37, 31 and 19 days, respectively. Considering the possibility of STEC intermittent long-term shedding, symptomatic and asymptomatic individuals should be excluded from the Institution until two consecutive stool cultures obtained at least 48 h apart, test negative.

Key words: Shiga toxin-producing Escherichia coli; Prolonged fecal shedding; Children day-care centers

RESUMEN

Excreción prolongada de Escherichia coli productor de toxina Shiga en niños que concurren a jardines maternales de Argentina. En el presente trabajo se describe la detección y el tiempo de excreción de Escherichia coli productor de toxina Shiga (STEC) O157 y no-O157 en casos sintomáticos y asintomáticos durante cuatro eventos ocurridos en jardines maternales de Argentina. En cada evento se identificaron los casos entre los niños, sus familiares y el personal del jardín. Los aislamientos fueron caracterizados por técnicas feno-genotípicas y de subtipificación. La excreción de STEC fue, en general, prolongada e intermitente. Cepas STEC O157:H7 (1^er evento); O26:H11 (2^do evento); O26:H11 (3^er evento) y O145:NM (4^to evento) fueron excretadas durante 23-30, 37, 31 y 19 días, respectivamente. Dadas las características de la excreción, no debe permitirse el reingreso a la institución de todo niño o adulto con infección por STEC, sintomático o asintomático, hasta no tener dos coprocultivos negativos sucesivos, con intervalos de 48 horas entre ellos.

Palabras claves: Escherichia coli productor de toxina Shiga; Excreción prolongada; Jardines maternales

Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 strains have been associated with outbreaks and sporadic cases of human disease, ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic uremic syndrome (HUS). STEC is transmitted to humans through contaminated food, water, and direct contact with infected persons or animals (1). Person-toperson transmission is well documented in day-care and extended-care facilities, most likely because of the low infection dose and the increased susceptibility of the individuals in these facilities (4). Shedding of E. coli O157 can be prolonged and intermittent (12). In contrast, little information about STEC non-O157 excretion is available. The aim of this study was to describe the detection and duration of fecal shedding of STEC O157 and non-O157 in symptomatic and asymptomatic cases during four events occurred among children in day-care centers. Rectal swabs were collected and routinely cultured for intestinal pathogens at the Regional Microbiological Laboratories. Sorbitol-MacConkey agar plates were sent to the National Reference Laboratory (NRL) for screening purposes. The STEC strains were isolated and characterized as described elsewhere (8, 10). The duration of STEC shedding was defined as the interval between the onset of diarrhea and the last positive sample followed by two consecutive negative stool cultures obtained at least 48 h apart.
1^st Event: In November 2002, a 12-month-old girl, attending a day-care center in Buenos Aires City, had bloody diarrhea associated with E. coli O157:H7. A study was conducted to determine the E. coli O157 spreading among 11 children, 3 staff members, and 6 household contacts. Only one positive-asymptomatic five-month-old boy, attending the same room, was identified. E. coli O157:H7 strains isolated from both children carried: stx_{2-O157-EDL933} + stx_{2c-O157-E32511}, LEE-encoded intimin ( eae) gene and enterohemolysin ( ehxA) genes, belonged to phage type (PT) 47, and showed the same profile by XbaI-pulsedfield gel electrophoresis ( XbaI-PFGE). The shedding was prolonged and intermittent in both children. The symptomatic child carried the organism for 23 days and the asymptomatic child for 30 days respectively.
2^nd Event: From October 15 to November 8, 2003, a gastrointestinal outbreak occurred at a day-care center in Mar del Plata City. Fourteen (17.5%) out of 80 children, and one of the clhildren's mother had diarrhea (3). One case developed HUS. A STEC O26:H11 strain in a 9-month-old boy with uncomplicated diarrhea, and a STEC O103:H2 strain in another 11-month-old boy were identified. Both isolates were positive for stx_{1-O157-EDL933}, eae and ehxA genes. The child with the STEC O26:H11 infection shed the pathogen for 37 days, whereas the child with the STEC O103:H2 infection had a shorter excretion because fecal samples taken at 15 and 29 days following the onset of symptoms were STEC-negative. Although STEC strains were isolated in two children, they were not associated as etiologic agents of the outbreak.
3_rd Event: Between January 23 and 31, 2004, an outbreak of bloody diarrhea occurred at a day-care center in Paraná City (5). Four (10%) out of 40 children presented bloody diarrhea and one case developed HUS. All children, 5 staff members, and six family contacts of cases were screened for STEC infection. E. coli O157:H7 was isolated from three bloody diarrhea cases and one asymptomatic child. The isolates were characterized as stx_{2-O157- EDL933} + stx_{2c-O157-E32511}, eae, ehxA, and PT4, and showed an indistinguishable XbaI-PFGE pattern. In another asymptomatic child, a PCR- stx₂/ rfbO157 positive sample was identified, without isolation. One asymptomatic child with E. coli O26:H11, stx_{1-O157-EDL933,} eae, ehxA infection continued shedding the organism for 31 days. All specimens from the staff and family contacts were negative.
4_th Event: On January 6, 2005, four HUS cases occurred at a day-care center in Rosario City. Stool samples from attendees, staff members of the Institution and family contacts of confirmed positive-STEC cases were collected and analyzed. At the NRL, 3 (5%) of 53 samples were confirmed as STEC-positive. An infection associated with STEC O145:NM, stx_{2-O157-EDL933}, eae, and ehxA was confirmed in a 12-month-old-boy and a 24- month-old-girl. An STEC ONT:HNT, Stx_{2d2-O91/b-B2F1}, eae, and ehxA was recovered from an asymptomatic teacher. The O145:NM positive-boy continued to shed the organism for 18 days, but during the fifth control, an association with a ONT:HNT STEC strain with the same genotypic profile as the teacher's strain, was detected. The O145:NM positive-girl and the ONT:HNT positiveteacher, shed the strains for 19 days. All the O145:NM strains showed the same XbaI-PFGE pattern. All specimens from family contacts were negative.
This report showed the long-term shedding of STEC O157 and non-O157 during day-care-associated outbreaks. Shah et al. reported a median duration of O157 shedding of 29 days (range, 11-57) during a day-careassociated outbreak (11). A study performed in Germany, revealed a median duration of O157 shedding of 13 days (range, 2-62) and 21 days (range, 5-124) in diarrhea and HUS patients, respectively (6). To our knowledge, this is one of the first reports of non-O157 long term shedding in humans. It is important to remark that E. coli O145:NM and O26:H11 have been associated with severe human disease worldwide and they are the second and third most common serotypes isolated in Argentina, respectively (9). Similarly to Gouveia et al. (4), we recommended that in the event of day-care center outbreaks, asymptomatic children and personnel should be tested because they may also be infected and shedding this pathogen despite the absence of symptoms. We also emphasize that symptomatic and asymptomatic children and staff members excreting STEC should not be allowed to return to the day-care facilities as long as they continue to shed this pathogen. Because of the possibility of STEC intermittent long-term shedding, the standard for no infectivity might require two negative stool samples collected at 2- day interval. The study by Belongia et al. (2) has shown that there was no evidence of person-to-person transmission at day-care facilities when children with diarrhea were allowed to return to the facility after two consecutive O157-negative stool cultures. Moreover, individuals with diarrhea illness should be excluded from the center until symptoms resolve whether or not a pathogen is identified. These four events emphasize the importance of prompt case reporting, as well as follow- up testing for the pathogen in stool culture. In this study, a multiplex-PCR for stx1 / stx 2 / rfbO157 gene detection, a sensitive and specific technique for screening of both O157 and non-O157 STEC strains, was included (7). Day-care centers are institutions where hygiene practices are difficult to ensure. It is important to consider that routine hand washing is one of the best ways to prevent the spread of infection among the children. Considering that the use of the swimming pool was identified as the only risk factor epidemiologically linked to the 3^rd event (OR, 10.0; 95% CI, 1.3-77.0) (5), efforts to educate pool owners and parents on the importance of proper pool maintenance with continuous chlorination, are necessary to improve safety in this type of activities. The management of these events highlighted both the long-term STEC shedding and the risks posed by child care facilities in the pathogen transmission. The knowledge of transmission routes and vehicles will allow professional health workers, teachers and parents to be educated on reducing risky behavior, which can decrease their risk for STEC infection.

Acknowledgements: This work was supported by grants from Fundación Alberto J. Roemmers (Argentina).

REFERENCES

1. Armstrong G, Hollingsworth J, Morris G. Emergin foodborne pathogens: Escherichia coli O157:H7 as a model of entry of new pathogen into the food supply of the developed world. Epidemiologic Reviews 1996; 18: 29-51.
2. Belongia E, Osterholm M, Soler J, Ammend D, Braun J. Transmission of Escherichia coli O157:H7 infection in Minnesota child day-care facilities. JAMA 1993; 269: 883-8.
3. Gomez D, Miliwebsky E, Silva A, Deza N, Zotta C, Cotella O, et al. Aislamiento de Escherichia coli productor de toxina Shiga durante un brote de gastroenteritis en un Jardín Maternal de la Ciudad de Mar del Plata. Rev Argent Microbiol 2005; 37:176-83.
4. Gouveia S, Proctor M, Lee M, Luchansky J. Genomic comparisons and Shiga toxin production among Escherichia coli O157:H7 from a day care center outbreak and sporadic cases in southeastern Wisconsin. J Clin Microbiol 1998; 36: 727-33.
5. Ilardo M, Agüero G, Chinen I, Miliwebsky E, Pedroni E, Sosa-Estani S, et al. Brote de diarrea por Escherichia coli O157:H7 en un Jardín Maternal. Paraná-Entre Ríos, Enero 2004. Congreso Latinoamericano y X Congreso Argentino de Microbiología, 2004, Resumen 643, Buenos Aires, Argentina.
6. Karch H, Russman H, Schmidt H. Long-term shedding and clonal turnover of enterohemorrhagic Escherichia coli O157 in diarrheal diseases. J Clin Microbiol 1995; 33: 1602-5.
7. Leotta GA, Chinen I, Epszteyn S, Miliwebsky E, Melamed IC, Motter M, et al. Validación de una técnica de PCR multiple para la detección de Escherichia coli productor de toxina Shiga. Rev Argent Microbiol 2005; 37: 1-10.
8. Rivas M, Caletti MG, Chinen I, Refi SM, Roldán CD, Chillemi G et al. Home-prepared hamburger and sporadic hemolytic uremic syndrome, Argentina. Emerg Infect Dis 2003; 9: 1184-6.
9. Rivas M. Control and prevention of Shiga toxin-producing Escherichia coli infections in humans and in the animal reservoir. II Simposio Internacional de Biotecnología, 2004, Conferencia C5, p. 5, Tucumán, Argentina.
10. Scheutz F, Beutin L, Pierard D, Smith R. Nomenclature of verocytotoxins. In: Garvey P, McDowell DA, editors. Verocytotoxigenic E. coli. Trumbull, Conneticut, USA. Food & Nutrition Press, 2001, p 447-52.
11. Shash S, Hoffman R, Shillam P, Wilson B. Prolonged fecal shedding of Escherichia coli O157:H7 during an outbreak at day care center. Clin Infect Dis 1996; 23:835-6.
12. Swerdlow DL, Griffin PM. Duration of faecal shedding of Escherichia coli O157:H7 among children in day-care centres. Lancet 1997; 349: 745-6.        [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]

Recibido: 18/09/06
Aceptado: 09/04/07