adherence factor (EAF) genes, and diarrhea showed that only EAF-positive (EAF+) E. coli strains belonging to the

Transcription

1 ~~~~~~~~ INFECrION AND IMMUNITY, Mar. 1993, p /93/ $02.00/0 Copyright 1993, American Society for Microbiology Enteropathogenic Escherichia coli (EPEC) serogroups have been identified as important agents of infant diarrhea in many developing countries (2, 3, 5, 6, 10, 11, 13, 14). More recently, only the following specific serotypes (classic serotypes) within these serogroups were recognized as belonging to the EPEC category: 026:H-, 026:H11, 055:H-, 055: H6, 055:H7, 086:H34, 0111ab:H-, 0111ab:H2, Olllab:., - FIG. 1. Attaching-Effacing Lesions and Intracellular Penetration in HeLa Cells and Human Duodenal Mucosa by Two Escherichia coli Strains Not Belonging to the Classical Enteropathogenic E. coli Serogroups M. Z. PEDROSO,* E. FREYMULLER, L. R. TRABULSI, AND T. A. T. GOMES Department of Microbiology, Immunology and Parasitology and Centre of Electron Microscopy, Escola Paulista de Medicina, Rua Botucatu, 862 V. Clementino, CEP 04023, Sdo Paulo, and Department of Microbiology, Instituto de Ciencias Biomedicas, Universidade de Saio Paulo, Sdo Paulo, Brazil Received 13 July 1992/Accepted 30 November 1992 In the present study, we compared two strains of serotypes 088:H25 and 0145:H45 with an enteropathogenic Escherichia coli (EPEC) adherence factor-positive (EAF+) strain of the classic enteropathogenic E. coli serotype 0111ab:H2 for their association with HeLa cells and with biopsies of human duodenal mucosa. Both strains not belonging to the classic EPEC serotype showed virulence properties similar to those of the serotype 0111ab:H2 strain, i.e., the production of attaching-effacing lesions and intracellular penetration in both systems. These virulence properties associated with the relatively high frequency at which the two serotypes had been detected in infant diarrhea in Sao Paulo, Brazil (T. A. T. Gomes, M. A. M. Vieira, I. K. Wachsmuth, P. A. Blake, and L. R. Trabulsi, J. Infect. Dis. 160: , 1989) allowed us to suggest that strains of serotypes 088:H25 and 0145:H45 should be included in the EAF+ EPEC category. Vol. 61, No. 3 adherence factor (EAF) genes, and diarrhea showed that only EAF-positive (EAF+) E. coli strains belonging to the classic serotypes were statistically associated with infant diarrhea (5). However, in that study, EAF+ E. coli strains not belonging to the classic EPEC serogroups were detected, and localized adherence (LA) production by these strains in HeLa cells was confirmed. Although as a group these Electron micrographs of HeLa cells after incubation for 7 h at 37 C with Olllab:H2 (A), 088:H25 (B), and 0145:H45 (C). Bacteria enclosed in membrane-bound vacuoles were seen in most cells (initial ratio, 4 x 102 bacterial cells per HeLa cell) (arrows). Magnifications: A, x8,000; B, x6,000; C, x3,125. H12, 0111ab:H21, 0114:H2, 0119:H6, 0125ac:H21, 0126: H-, 0126:H27, 0127:H-, 0127:H6, 0128ab:H2, 0128ab: H7, 0142:H6, and 0158:H23 (5). In Sao Paulo, Brazil, a study conducted to examine the interrelationships of EPEC serotypes and serogroups, EPEC * Corresponding author. serotypes were not associated with diarrhea, at least three of them (088:H25, 0145:H45, and 0153:H7) were individually found at frequencies that were higher than the frequency of EAF+ strains of some classic EPEC serotypes. However, other virulence mechanisms presently attributed to EAF+ EPEC serotypes, such as attaching-effacing (ANE) lesions (8) and intracellular penetration, should be searched for in these non-epec serotypes to better understand their role in infant diarrhea. 1152

2 VOL. 61, 1993 NOTES 1153 FIG. 2. SEM views of surfaces of human duodenal biopsies infected with 0111ab:H2 (A), 088:H25 (B), and 0145:H45 (C) for 8 h at 37 C. Bacteria can be seen (arrows) in contact with each other and attached to the epithelial surfaces. The surrounding epithelial surfaces appear unaltered. Magnifications: A, x6,540; B, x6,250; C, x7,500. In the present study, we compared two E. coli EAF+ strains of serotypes 088:H25 and 0145:H45 with an EAF+ strain of the classic EPEC serotype Olllab:H2 for their association with HeLa cells and with biopsies of human duodenal mucosa. We found that both strains not belonging to the classic EPEC serogroups had virulence properties similar to those of the serotype Olllab:H2 strain, i.e., the production of A/E lesions and intracellular penetration in both systems. The three LA-positive EAF+ E. coli strains studied were isolated from infant diarrheic feces in which no other recognized enteropathogens had been identified (5) and were stored at -70 C. The eae probe (7) was used to detect DNA sequences associated with A/E lesions. All three strains reacted with this probe. For the HeLa cell and duodenal mucosa assays, bacterial strains were grown overnight in 3 ml of tryptic soy broth (TSB). E. coli K-12 HB101 was used as a negative control. Invasion of HeLa cells was detected by the method of Miliotis et al. (12). After incubation for 7 h, infected HeLa cells were washed in phosphate-buffered saline (PBS) and fixed in 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (ph 7.2) for 30 min, gently scraped, and washed in 9% saccharose (Sigma) in 0.05 M cacodylate buffer (ph 7.2). Cell pellets were placed in 1% agarose (Sigma), and small blocks were cut and prepared for transmission electron microscopy (TEM). Cultured human intestinal mucosa assays were performed as described by Knutton et al. (8) with some modifications. Duodenal mucosa biopsies were taken from adult patients undergoing endoscopy after informed consent had been obtained. Biopsies were taken immediately to the laboratory after being placed in ice-cold NCTC 135 medium (Sigma) with 200 plg of gentamicin per ml and washed in medium without antibiotic. Two biopsy samples from different patients were placed on sterile filters (AP15; Millipore) in plastic petri dishes (35 by 10 mm; Corning). After the addition of 4.8 ml of the organ culture medium described by Embaye et al. (4) and containing 1% D-mannose (Sigma), the dishes were maintained for 15 min at 37 C in an incubator with 95% 02-5% CO2. Two hundred microliters of bacterial culture was added to the biopsy samples, and the mixtures were incubated for 2 h at 37 C with gentle agitation in an incubator with 95% 02-5% CO2. Biopsy samples were then washed six times in NCTC 135 medium and incubated with 5 ml of organ culture medium without antibiotic for an additional 6 h and with antibiotic for an additional 10 or 16 h under the conditions described above. After these incubations, the biopsy samples were washed and prepared for FIG. 3. TEM views of human duodenal biopsies infected with 0111ab:H2 (A), 088:H25 (B), and 0145:H45 (C) for 8 h at 37 C. Bacilli can be seen closely adherent to enterocyte membranes (arrowheads). The characteristic effacement of microvilli (mv), membrane cupping, and pedestal formation at the sites of attachment can be seen (stars). Magnifications: A, x 12,000; B, x 12,500; C, x 12,115.

3 1154 NOTES ji INFECT. IMMUN. I, 4sgW.,'~ A FIG. 4. TEM views of human duodenal biopsies infected with 0111ab:H2 (A), 088:H25 (B), and 0145:H45 (C) for 12 h at 37 C. Bacterial cells can be seen undergoing internalization (arrows). Magnifications: A, x 16,578; B, x 16,428; C, x 17,142. TEM. For scanning electron microscopy (SEM), medium without antibiotic was always used. The HeLa cell invasion assays performed with the three strains showed a few bacterial cells undergoing internalization and others enclosed in membrane-bound vacuoles (Fig. 1). E. coli K-12 HB101 was not associated with HeLa cells. SEM of duodenal mucosa infected by 088:H25, Olllab: H2, and 0145:H45 demonstrated that the mucosal surface was colonized by bacteria during 8 h of incubation (Fig. 2). For the same period, TEM demonstrated that the three E. coli strains caused characteristic A/E lesions, i.e., brush border effacement, cup and pedestal formation with bacterial and enterocyte membranes being in close proximity, and elongated microvilli located at sites of bacterial attachment (Fig. 3). After 12 h of incubation, a few bacterial cells of the three serotypes were observed to be undergoing internalization (Fig. 4). After 18 h of incubation, intracellular penetration by the three serotypes studied and bacteria free in the cytoplasm of enterocytes could be observed (Fig. 5). E. coli K-12 HB101 was not found in duodenal biopsy samples. In a case control study, Echeverria et al. (3) reported the occurrence of EAF+ E. coli strains in 3.3% of infants with diarrhea and 1.9% of control infants. Although these strains did not show a statistical association with diarrhea, they were able to produce A/E lesions, as identified in the fluorescent-actin staining assay (9). More recently, Albert et al. (1) demonstrated that three EAF+ non-epec strains of serotypes 02:H2, 02:H25, and 015:H2 and isolated from infant diarrhea caused A/E lesions in HeLa cells, as detected by use of the fluorescent-actin staining assay and rabbit ileal segments. In this study, besides causing A/E lesions, the non-epec serotype 088:H25 and 0145:H45 strains were able to invade HeLa cells and human duodenal mucosa. In a previous study conducted in our laboratory, strains of serotype 088:H25 were isolated from six patients with

4 VOL. 61, 1993 NOTES 1155 tw, I Downloaded from FIG. 5. Electron micrographs of sections of human duodenal biopsies infected with O111ab:H2 (A), 088:H25 (B), and 0145:H45 (C) after 18 h of incubation at 37'C. Intracellular penetration by the three serotypes studied and bacteria free in the cytoplasm can be seen (arrows). Magnifications: A, x 12,125; B, x 12,000; C, x 11,428. diarrhea but not from controls, this difference being statistically significant (5). Morever, strains of serotype 0145:H45, although not statistically associated with diarrhea (three patients and one control), occurred at frequencies similar to or higher than those observed for other recognized EAF+ EPEC serotypes, such as 055:H-, 086:H34, 0127:H6, and 0142:H6. The relatively high frequencies at which serotypes 088: H25 and 0145:H45 occurred in infant diarrhea in Sao Paulo, Brazil (5), and the demonstration of specific, EPEC-related virulence factors in a strain of each of these two serotypes suggest that when similar behavior is observed for other strains of these serotypes, these strains should be included in the EAF+ EPEC category. This work was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (grant 89/2341-9). We thank James B. Kaper for supplying us with the eae probe and Marcia Araguth for technical assistance with electron microscopy. We also thank Angelo P. Ferrari for providing human intestinal tissue. REFERENCES 1. Albert, M. J., K. Alam, M. Ansaruzzaman, J. Montanaro, M. Islam, S. M. Faruque, K. Haider, K. Bettelheim, and S. Tzipori Localized adherence and attaching-effacing properties of nonpathogenic serotypes of Escherichia coli. Infect. Immun. 59: Echeverria, P., F. 0rskov, I. 0rskov, S. Knutton, F. Scheutz, J. E. Brown, and U. Lexomboon Attaching and effacing enteropathogenic Escherichia coli as a cause of infantile diarrhea in Bangkok. J. Infect. Dis. 164: Echeverria, P., D. N. Taylor, U. Lexomboon, et al Case-control study of endemic diarrheal disease in Thai children. J. Infect. Dis. 159: Embaye, H., R. M. Batt, J. R. Saunders, B. Getty, and C. A. Hart Interaction of enteropathogenic Escherichia coli 0111 with rabbit intestinal mucosa in vitro. Gastroenterology 96: Gomes, T. A. T., M. A. M. Vieira, I. K. Wachsmuth, P. A. on March 8, 2019 by guest

5 1156 NOTES INFECT. IMMUN. Blake, and L. R. Trabulsi Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhea in Sao Paulo, Brazil. J. Infect. Dis. 160: Huilan, S., L. G. Zhen, M. M. Mathan, M. M. Mathew, J. Olarte, R. Espejo, K. M. V. Maung, M. A. Ghafoor, M. A. Khan, Z. Sami, and R. G. Sutton Etiology of acute diarrhea among children in developing countries: a multicentre study in five countries. Bull. W.H.O. 69: Jerse, A. E., J. Yu, B. D. Tall, and J. B. Kaper A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87: Knutton, S., D. R. Lloyd, and A. S. McNeish Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect. Immun. 55: Knutton, S., A. D. Phillips, H. R. Smith, R. J. Gross, R. Shaw, P. Watson, and E. Price Screening for enteropathogenic Escherichia coli in infants with diarrhea by the fluorescent-actin staining test. Infect. Immun. 59: Levine, M. M., and R. Edelman Enteropathogenic Escherichia coli of classical serotypes associated with infant diarrhea: epidemiology and pathogenesis. Epidemiol. Rev. 6: Levine, M. M., J. P. Nataro, H. Karch, M. M. Baldini, J. B. Kaper, R. E. Black, M. L. Clements, and A. D. O'Brien The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor. J. Infect. Dis. 152: Miliotis, M. D., H. J. Koornhof, and J. I. Phillips Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model. Infect. Immun. 57: Toledo, M. R. F., M. C. B. Alvariza, J. Murahovschi, S. R. T. S. Ramos, and L. R. Trabulsi Enteropathogenic Escherichia coli serotypes and endemic diarrhea in infants. Infect. Immun. 39: Trabulsi, L. R., M. R. F. Toledo, J. Murahovschi, U. Fagundes Neto, and J. A. N. Candeias Epidemiology of diarrhoeal diseases in South America, p In S. Tzipori (ed.), Infectious diarrhoea in the young. Elsevier Biomedical Press, Amsterdam. Downloaded from on March 8, 2019 by guest